Method of synthesis of peptidyl aldehydes

ABSTRACT

This invention provides solution-phase and solid-phase methods for the synthesis of peptidyl argininals and to  novel! reagents useful therein.

CROSS REFERENCE TO RELATED APPLICATION

This application is a Continuation-in-Part of U.S. Ser. No. 08/261,380,filed Jun. 17, 1994 now U.S. Pat. No. 5,514,177, the disclosure of whichis incorporated herein by reference.

TECHNICAL FIELD

This invention relates to solution-phase and solid-phase methods for thesynthesis of peptidyl argininals and to novel reagents useful therein.Using the disclosed methods and reagents of the present invention,peptidyl argininals can be rapidly and efficiently produced. Thepeptidyl argininals are useful as enzyme inhibitors, in vitro diagnosticagents and in vivo pharmaceutical agents.

BACKGROUND

The trypsin sub-family of serine proteases (referred to as thetrypsin-like serine proteases) is composed of proteases which hydrolyzepeptide bonds that follow an arginine or lysine residue. These proteasesplay an important physiological role in digestion, coagulation,fibrinolysis, blood pressure regulation, fertility, and inflammation."Design of Enzyme Inhibitors as Drugs", Oxford Science Publications,(Edits. Sandler, M., Smith, H. J. 1989). Selective inhibitors oftrypsin-like serine proteases are thought to be useful as drugs forintervention into many disease states in which the involvement of theseproteases has been implicated.

Peptide analogs which utilize the catalytic mechanism of an enzyme (e.g.transition-state inhibitors) have been suggested as inhibitors of thetrypsin-like serine proteases. The catalytic mechanism of theseproteinases is thought to involve the attack of the active-site serineon the carbonyl bearing the scissile amide bond of the substrate, togive a tetrahedral intermediate which subsequently results in peptidebond cleave. It has been reported that peptide analogs which are stablemimics of this tetrahedral intermediate (i.e., transition-state analogs)can be selective enzyme inhibitors. Delbaere, L. T. J., Brayer, G. D.,J. Mol. Biol. 183:89-103, 1985 and "Proteases and Biological Control",Cold Spring Harbor Laboratory Press, pp. 429-454 (Edits. Aoyagi, T. andUmezawa, H. 1975). Selective transition-state inhibitors of thetrypsin-like serine proteases may therefore be useful as drugs forintervention into many disease states in which the involvement of theseproteases has been implicated.

One candidate group of transition-state inhibitors which may beparticularly useful are the peptide analogs which have an aldehyde groupon the C-terminus of the peptide analog. Peptide aldehydes wereinitially discovered as natural products produced by a number ofactinomycete strains. Some derivatives of natural products have beenreported to be selective inhibitors of various types of serine andcysteine proteinases. Aoyagi, T., Supra. For example, the peptide,alaninal elastatinal, was reported to be a potent elastase inhibitor,but not an inhibitor of trypsin or the trypsin-like serine proteases.Hassall, C. H. et al., FEBS Lett., 183:201-205 (1985). Elastaseinhibitors are of interest in the treatment of diseases such asemphysema and synthetic peptide aldehydes have been reported to beexcellent inhibitors of human leukocyte elastase. Sandler, M., Smith, H.J., Supra. It has been reported that the selectivity of these naturallyoccurring analogs has been enhanced by modifying the peptide sequence.Bajusz, S. et al., J. Med. Chem. 33:1729-1735 (1990); and McConnell, R.M. et al., J. Med. Chem. 33:86-93 (1990).

The peptidyl argininal, leupeptin (Acetyl-L-Leu-L-Leu-L-Arg-al), hasbeen reported to be a selective inhibitor of trypsin-like serineproteases. "Structures and activities of protease inhibitors ofmicrobial origin", Proteases and Biological Control, Cold Spring HarborLaboratory Press, pp. 429-454 (Edits. Aoyagi, T., Umezawa, H. 1975).Leupeptin, along with its naturally occurring variants and syntheticanalogs, have been reported to be potent inhibitors of severaltrypsin-like serine proteases in the coagulation cascade.

The peptide argininal, D-Phe-L-Pro-L-Arg-al, and analogs thereof, havebeen reported to show a marked selectivity for particular coagulationfactors. For example, one such analog (N-methyl-D-Phe-Pro-Arg-al) hasbeen developed as a thrombin inhibitor and is reported to havesignificant in vivo anticoagulant activity. U.S. Pat. Nos. 4,316,889(1982), 4,399,065 (1983), 4,478,745 (1984), 4,346,078 (1982), and4,708,039 (1987).

A major problem in medical research directed to the use of peptidylaldehydes as potential drugs for intervention into many disease statesin which trypsin-like serine proteases have been implicated has been thedifficulty in synthesizing the peptidyl argininals. Thoughsolution-phase methods for their synthesis have been reported, theirsynthesis remains a labor-intensive and time-consuming process.

Three methods for the solution-phase synthesis of peptidyl argininals(Arg-al), each using a different intermediate, have been reported.

The use of L-Leu-L-Arg-al dibutylacetal as an intermediate has beenreported in the synthesis of more than 30 peptidyl argininals. Inparticular, L-Leu-L-Arg-al was reported to be prepared by thermolysindigestion of leupeptin (acetyl-L-Leu-L-Leu-L-Arg-al), transformation ofthe digestion product to a racemic dibutyl acetal(L-Leu-D,L-Arg-dibutylacetal), followed by separation of thediastereomers. Saino, T et al., Chem. Pharm. Bull., 30(7):2319 (1982);T. Saino et al., J. Antibiotics, 41:220 (1988).

The use of the N^(w) -carbobenzyloxy-arginine lactam as an intermediatein the synthesis of peptidyl argininals has been reported. The lactamwas reported to be coupled to a variety of peptides in good to highyield. The resulting peptidyl-N^(w) -carbobenzyloxy-arginine lactam wasreduced with LiAlH₄ to form the peptidyl-N^(w)-carbobenzyloxy-argininal, and subsequently hydrogenated to give thepeptidyl argininal. Basjusz, S. et al, J. Med. Chem., 33:1729 (1990);Shuman, R. T. et al., J. Med. Chem., 36:314 (1993); Balasubramanian, N.et al., J. Med. Chem., 36:300 (1993).

The use of semicarbazone intermediates has been reported in thesynthesis of peptidyl argininals. The unsubstituted semicarbazone, N^(g)-nitro-L-argininal semicarbazone, was used as an intermediate in thesynthesis of peptidyl argininals. McConnell, R. M. et al., J. Med Chem.,35:86 (1990); R. M. McConnell, J. L. York, D. Frizzell, C. Ezell, J. MedChem., 36, 1084-1089 (1993). N^(g) -nitro-L-argininalsemicarbazonyl-4-methylcyclohexane carboxylic acid was reported as anintermediate in the preparation of peptide aldehydes by a solid phasemethod. Murphy, A. M. et al., J. Am. Chem. Soc., 114:3156 (1992); andWebb, T. R., U.S. Pat. No. 5,283,293 (Feb. 1, 1994). N^(g)-nitro-L-argininal semicarbazonyl-4-diphenylmethane was reported as anintermediate for the solution-phase synthesis of peptidyl argininals.Brunck, T. K. et al., WO 93/14779 (1993).

SUMMARY OF THE INVENTION

In one aspect, the present invention is directed to novel compoundsuseful for the solution-phase synthesis of peptidyl argininals. Thesecompounds have the formula:

wherein ##STR1## wherein

R₁ is selected from the group consisting of hydrogen, benzyloxycarbonyl,isonicotinyloxycarbonyl, 2-chlorobenzyloxycarbonyl,4-methoxybenzyloxycarbonyl, t-butoxycarbonyl, t-amyloxycarbonyl,isobornyloxycarbonyl, adamantyloxycarbonyl,2-(4-biphenyl)-2-propyloxycarbonyl, 9-fluorenylmethoxycarbonyl andmethylsulfonylethoxycarbonyl;

R₂ is selected from the group consisting of alkyl of 1 to about 12carbon atoms and aralkyl of about 7 to about 15 carbon atoms, either ofwhich can be substituted with a hydroxy, and --CO--Y, wherein Y ishydroxy, alkoxy of 1 to about 12 carbon atoms, aralkoxy of about 7 toabout 15 carbon atoms O-polymeric support or NH-polymeric support;

R3 is selected from the group consisting of hydrogen, Fmoc, nitro,benzyloxycarbonyl, t-butoxycarbonyl and adamantyloxycarbonyl; and

R₄ is selected from the group consisting of hydrogen, alkyl of 1 toabout 12 carbon atoms, aryl of about 6 to about 14 carbon atoms andaralkyl of about 7 to about 15 carbon atoms; and salts thereof.

In another aspect, the present invention is directed to salts of thecompounds of the present invention.

In yet another aspect, the present invention is directed to methods ofpreparing peptidyl argininals, which comprises:

(a) reacting a first intermediate having the formula: ##STR2## whereinR₅ is selected from the group consisting of benzyloxycarbonyl,isonicotinyloxycarbonyl, 2-chlorobenzyloxycarbonyl,4-methoxybenzyloxycarbonyl, t-butoxycarbonyl, t-amyloxycarbonyl,isobornyloxycarbonyl, adamantyloxycarbonyl,2-(4-biphenyl)-2-propyloxycarbonyl, 9-fluorenylmethoxycarbonyl andmethylsufonylethoxycarbonyl;

R₆ is selected from the group consisting of alkyl of 1 to about 12carbon atoms and aralkyl of about 7 to about 15 carbon atoms, either ofwhich can be substituted with a hydroxy, and --CO--Y, wherein Y ishydroxy, alkoxy of 1 to about 12 carbon atoms or aralkoxy of about 7 toabout 15 carbon atoms, O-polymeric support or NH-polymeric support;

R7 is selected from the group consisting of hydrogen, Fmoc, nitro,benzyloxycarbonyl, t-butoxycarbonyl and adamantyloxycarbonyl; and

R₈ is selected from the group consisting of hydrogen, alkyl of 1 toabout 12 carbon atoms, aryl of about 6 to about 14 carbon atoms, andaralkyl of about 7 to about 15 carbon atoms;

with a R₅ removing reagent which chemically removes the R₅ group fromsaid first intermediate to give a second intermediate of the formula:##STR3## (b) chemically coupling to the second intermediate of step (a),a protected amino acid, a protected amino acid analog or a protectedpeptide of about 2 to about 30 amino acids, amino acid analogs, or acombination of amino acids and amino acid analogs, using a couplingreagent to give a third intermediate having the formula: ##STR4##wherein X is a protecting group,

k is an integer from 1 to 30, and

AA₁ -AA₂ . . . AA_(k) is an amino acid, amino acid analog or peptidecomprised of k amino acids, amino acid analogs or a combination of aminoacids and amino acid analogs;

(c) reacting the third intermediate with a R₇ removing reagent, when R₇is not already hydrogen, which chemically removes the R₇ group to give afourth intermediate having the formula: ##STR5## (d) reacting the fourthintermediate with a hydrolyzing reagent which comprises an aqueous acidto chemically hydrolyze said fourth intermediate to give said peptidylargininal.

Thus, provided are methods of making peptidyl argininals comprising thesteps of:

(a) preparing a first intermediate having the formula: ##STR6## whereinR₅ is selected from the group consisting of benzyloxycarbonyl,isonicotinyloxycarbonyl, 2-chlorobenzyloxycarbonyl,4-methoxybenzyloxycarbonyl, t-butoxycarbonyl, t-amyloxycarbonyl,isobornyloxycarbonyl, adamantyloxycarbonyl,2-(4-biphenyl)-2-propyloxycarbonyl, 9-fluorenylmethoxycarbonyl andmethylsulfonylethoxycarbonyl;

R₆ is selected from the group consisting of alkyl of 1 to about 12carbon atoms and aralkyl of about 7 to about 15 carbon atoms either ofwhich can be substituted with a hydroxy and --CO--Y, wherein Y iscarboxy, alkoxy of 1 to about 12 carbon atoms, aralkoxy of about 7 toabout 15 carbon atoms, O-polymeric support or NH-polymeric support;

R₇ is selected from the group consisting of hydrogen, Fmoc, nitro,benzyloxycarbonyl, t-butoxycarbonyl and adamantyloxycarbonyl; and

R₈ is selected from the group consisting of hydrogen, alkyl of 1 toabout 12 carbon atoms, aryl of about 6 to about 14 carbon atoms, andaralkyl of about 7 to about 15 carbon atoms;

(b) chemically removing the R₅ group from said first intermediate togive a second intermediate;

(c) chemically coupling to said second intermediate having its R₅ groupremoved, a protected amino acid, protected amino acid analog orprotected peptide comprised of about 2 to about 30 amino acids, aminoacid analogs, or a combination of amino acids and amino acid analogs, togive a third intermediate having the formula: ##STR7## wherein x is aprotecting group,

k is an integer from 1 to 30, and

AA₁ -AA₂ . . . AA_(k) is an amino acid, amino acid analog or peptidecomprised of k amino acids, amino acid analogs or a combination of aminoacids and amino acid analogs;

(d) chemically removing the R₇ group from said third intermediate, whenR₇ is not hydrogen, to give a fourth intermediate having the formula:##STR8## (e) chemically hydrolyzing said fourth intermediate in a liquidcomprising an aqueous acid to give the product peptide argininal.

In yet another aspect, the present invention is directed to peptidylargininals made by the disclosed methods.

Definitions

In accordance with the present invention and as used herein, thefollowing terms are defined to have the following meanings, unlessexplicitly stated otherwise.

The term "alkyl" refers to saturated aliphatic groups includingstraight-chain, branched-chain and cyclic groups.

The term "alkoxy" refers to a group having the formula, R--O--, whereinR is an alkyl group.

The term "aryl" refers to aromatic groups which have at least one ringhaving a conjugated pi electron system and includes carbocyclic aryl,heterocyclic aryl and biaryl groups, all of which may be optionallysubstituted.

The term "aryloxy" refers to a group having the formula, R--O--, whereinR is an aryl group.

The term "aralkyl" refers to an alkyl group substituted with an arylgroup. Suitable aralkyl groups include benzyl, picolyl, and the like,all of which may be optionally substituted.

The term "aralkoxy" refers to a group having the formula, R--O--,wherein R is an aralkyl group.

The term "amino acid" refers to both natural amino acids, unnaturalamino acids, and amino acid analogs, all in their D and L stereoisomersif their structures allow such stereoisomeric forms. Natural amino acidsinclude alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid(Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine(Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys),methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser),threonine (Thr), tryptophan (Trp), tyrosine (Tyr) and valine (Val).Unnatural amino acids include, but are not limited toazetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid,beta-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyricacid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyricacid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4 diaminoisobutyricacid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid,N-ethylglycine, N-ethylasparagine, hydroxylysine, allo-hydroxylysine,3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine,N-methylglycine, N-methylisoleucine, N-methylvaline, norvaline,norleucine, ornithine and pipecolic acid. Amino acid analogs include thenatural and unnatural amino acids which are chemically blocked,reversibly or irreversibly, or modified on their N-terminal amino groupor their side-chain groups, example, example, methionine sulfoxide,methionine sulfone, S-(carboxymethyl)-cysteine,S-(carboxymethyl)-cysteine sulfoxide and S-(carboxymethyl)-cysteinesulfone.

The term "amino acid analog" refers to an amino acid wherein theN-terminal amino group, C-terminal carboxy group or side chain group hasbeen chemically blocked or modified to another functional group.

The term "amino acid residue" refers to radicals having the structure:(1) --HN--R--C(O)--, wherein R typically is --CH(R')--, wherein R' is Hor a carbon containing substituent; or (2) ##STR9## wherein p is 1, 2 or3 representing the azetidinecarboxylic acid, proline or pipecolic acidresidues, respectively.

The term "L-argininal" refers to L-arginine in which the carboxy grouphas been replaced with an aldehyde group. L-argininal has the formula:##STR10##

The term "D-argininal" refers to D-arginine in which the carboxy grouphas been replaced with an aldehyde group. D-argininal has the formula:##STR11##

The term "non-adverse conditions" describes conditions of reaction orsynthesis which do not substantially adversely affect the skeleton ofthe peptide analog and/or its amino acid (and/or amino acid analog)components. One skilled in the art can readily identify functionalities,coupling procedures, deprotection procedures and cleavage conditionswhich meet these criteria.

The term "peptide" refers to oligomers of amino acids which are linkedby peptide bonds. The nomenclature used to define the peptides is thatspecified by Schroder & Lubke, "The Peptides," Academic Press (1965),wherein in accordance with conventional representation the amino groupat the N-terminus appears to the left and the carboxyl group at theC-terminus to the right.

The term "peptidyl argininal" refers to a peptide in which theC-terminal amino acid is either L-argininal or D-argininal.

The term "polymeric support" refers to a solid support to which aminoacids and amino acid derivatives can be coupled.

In addition, the following abbreviations stand for the following:

"N-Boc-N^(g) -nitro-L-arginine" refers to the compound which has theformula: ##STR12##

"D-Arg-al" refers to D-argininal.

"Boc" refers to t-butoxycarbonyl.

"BOP" refers tobenzotriazol-1-yloxy-tris-(dimethylamino)-phosphonium-hexafluorophosphate.

"Brine" means an aqueous saturated solution of sodium chloride.

"DCC" refers to 1,3-dicyclohexylcarbodiimide.

"DMF" refers to dimethylformamide. "EDC" refers toethyl-3-(3-dimethylamino)propylcarbodiimide hydrochloride salt.

"Fmoc" refers to 9-fluorenylmethoxycarbonyl.

"HBTU" refers to 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate.

"HCl" refers to hydrochloric acid.

"HF" refers to hydrofluoric acid.

"HOBt" refers to 1-hydroxybenzotriazole monohydrate.

"2-PrPen" refers to 2-propylpentanoyl.

"LiAlH₄ " refers to lithium aluminum hydride.

"LiAlH₂ (OEt)₂ refers to lithium aluminum dihydride diethoxide.

"MBHA" refers to 4-methylbenzhydrylamine resin.

"TBTU" refers to 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the reaction scheme for preparation of a compound of thepresent invention, N^(g) -nitro-L-argininal ethyl cyclol. In this FIG.,(i)-(iv) are defined as: i) isobutyl chloroformate, 1-methylpiperidine,tetrahydrofuran; iia) isobutyl chloroformate, 1-methylpiperidine,O,N-dimethylhydroxylamine, HCl salt, tetrahydrofuran; LiAlH₄,tetrahydrofuran; IIb) LiAlH₂ (OCH₂ CH₃)2, tetrahydrofuran; iii)concentrated HCl, ethanol; and iv) anhydrous acid, ethanol.

FIG. 2 depicts the reaction scheme for preparation of a compound of thepresent invention, N^(g) -benzyloxycarbonyl-L-argininal ethyl cyclol. Inthis FIG., (i)-(v) are defined as: i) benzyl chloroformate, aqueoussodium hydroxide; ii) isobutyl chloroformate, N-methylmorpholine,triethylamine, tetrahydrofuran; iii) LiAlH₄, tetrahydrofuran; iv)concentrated HCl, ethanol; v) anhydrous HCl, ethanol.

FIG. 3 depicts a method of making peptidyl argininals using thecompounds of the present invention. In this FIG., (i)-(iv) are definedas: i) trifluoroacetic acid in dichloromethane or anhydrous HCl inabsolute ethanol; ii) HBTU, HOBt, N-methylmorpholine, acetonitrile; iii)H₂, 10% palladium on carbon, ethanol, acetic acid, water; iv) HPF₆,aqueous acentonitrile.

DETAILED DESCRIPTION OF THE INVENTION

1. Preferred Compounds

In one aspect, the present invention is directed to compounds which areuseful as intermediates for the synthesis of peptidyl aldehydes. Thesecompounds have the formula: ##STR13## wherein R₁ R₂, R₃ and R₄ are aspreviously defined herein.

The preferred compounds of the present invention include those whereinR₄ is hydrogen or alkyl of 1 to about 12 carbon atoms. Suitable alkylsfor R₄ include methyl, ethyl, 1-propyl, 2-methyl-1-propyl,2,2-dimethyl-1propyl, 2-propyl, 2methyl-2-propyl, 1-butyl, 2-butyl,3-butyl, 3-methyl-1-butyl, 1-pentyl, cyclopentyl, 1-hexyl,cyclopentylmethyl, cyclohexyl, cyclohexylmethyl, 1heptyl, 4-heptyl,octyl, nonanyl, dodecanyl, adamantyl or adamantylmethyl. Especiallypreferred compounds include those wherein R₄ is hydrogen, methyl, ethylor propyl. More especially preferred compounds include those wherein R₄is hydrogen.

The preferred compounds of the present invention include those whereinR₃ is Fmoc, nitro, benzyloxycarbonyl, t-butoxycarbonyl,adamantyloxycarbonyl, 2,2,5,7,8pentamethylchroman-6sulfonyl,4methoxy-2,3,6-trimethylbenzenesulfonyl and 4-methylbenzenesulfonyl.

In one aspect, the preferred compounds of the present invention includethose wherein R₃ is nitro or benzyloxycarbonyl. In this case, thepreferred compounds include those wherein R₁ is hydrogen,t-butoxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl,adamantyloxycarbonyl, 4methoxybenzyloxycarbonyl,2-(4biphenyl)-2-propyloxycarbonyl, or Fmoc. More especially preferredcompounds include those wherein R₁ is hydrogen or t-butoxycarbonyl.Especially preferred compounds include those wherein R₃ is nitro and R₁is hydrogen or t-butoxycarbonyl.

In another aspect, the preferred compounds of the present inventioninclude those wherein R₃ is t-butoxycarbonyl or adamantyloxycarbonyl. Inthis case, the preferred compounds include those wherein R₁ is hydrogen,benzyloxycarbonyl, isonicotinyloxycarbonyl, 2chlorobenzyloxycarbonyl,9fluorenylmethoxycarbonyl or methylsulfonylethoxycarbonyl. Especiallypreferred compounds include those wherein R₁ is hydrogen orbenzyloxycarbonyl. More especially preferred compounds include thosewherein R₃ is t-butoxycarbonyl and R₁ is hydrogen, benzyloxycarbonyl.

The preferred compounds of the present invention include those whereinR₂ is alkyl of 1 to about 12 carbon atoms optionally substituted with--CO--Y. Suitable alkyls for R₂ include methyl, ethyl, 1-propyl,2-methyl-1-propyl, 2,2-dimethyl-1-propyl, 2-propyl, 2-methyl-2-propyl,1-butyl, 2-butyl, 3-butyl, 3-methy-1-butyl, 1-pentyl, cyclopentyl,1-hexyl, cyclopentylmethyl, cyclohexyl, cyclohexylmethyl, 1-heptyl,4-heptyl, octyl, nonanyl, dodecanyl, adamantyl or adamantylmethyl.Especially preferred compounds include those wherein R₂ is methyl,ethyl, propyl or isopropyl. More especially preferred compounds includethose wherein R₂ is ethyl.

Preferred compounds of the present invention also include those whereinR₃ is Fmoc and R₂ is substituted with --CO--Y, wherein Y is O-polymericsupport or NH-polymeric support. Suitable polymeric supports includeaminomethylated polystyrene resin, p-benzyloxybenzyl alcohol resin,Merrifield resin, Rink amide resin and MBHA resin. Preferred polymericsupports include Merrifield resin and MBHA resin. In this case, thepreferred compounds include those wherein R₁ is hydrogen,t-butoxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl,adamantyloxycarbonyl, 4-methoxybenzyloxycarbonyl, or2-(4-biphenyl)-2-propyloxycarbonyl. More especially preferred compoundsinclude those wherein R₁ is hydrogen or t-butoxycarbonyl. Especiallypreferred compounds include those wherein R₃ is Fmoc and R₁ is hydrogenor t-butoxycarbonyl.

Certain preferred compounds of the present invention include: ##STR14##

In another aspect, the present invention is directed to salts of thecompounds of formula (i). "Salt" includes within its definition, saltsof the compounds of the present invention derived from the combinationof such compounds and an organic or inorganic acid. In practice, the useof the salt form amounts to use of the base form. The compounds of thepresent invention are useful in both free base and salt form, with bothforms being considered as being within the scope of the presentinvention. These salts include acid addition salts, for example, saltsof hydrochloric acid, acetic acid, trifluoroacetic acid and othersuitable acid addition salts.

2. Preparation of Preferred Compounds.

The compounds of the present invention are synthesized by solution-phasemethods or by solid-phase methods.

Many of the starting materials used in the syntheses are readilyavailable from chemical vendors such as Aldrich, Sigma, NovaBiochemicals, Bachem Biosciences, Inc. and the like.

FIG. 1 illustrates preferred routes for the solution-phase synthesis ofthe compounds of the present invention, N-alpha-t-butoxycarbonyl-N^(g)-nitro-L-argininal ethyl cyclol and N^(g) -nitro-L-argininal ethylcyclol. The latter may be prepared either as its trifluoroacetate orhydrochloride salt. Further details for the synthesis of these compoundsare disclosed in Example 1 through 5.

FIG. 2 illustrates a preferred route for the solution-phase synthesis oftwo more of the compounds of the present invention,N-alpha-t-butoxycarbonyl-N^(g) -benzyloxycarbonyl-L-argininal ethylcyclol and N^(g) -benzyloxycarbonyl-L-argininal ethyl cyclol. The lattermay be prepared as its hydrochloride salt. Further details for thesynthesis of these compounds are disclosed in Example 24 through 29.

As exemplified in FIGS. 1 and 2, certain protected argininals (A3 ofFIG. 1 and B4 of FIG. 2) may be converted to the compounds of thepresent invention by cyclization. FIG. 1 provides a preferred reactionscheme for cyclization of A3 to give A4. Likewise, FIG. 2 provides apreferred reaction scheme for cyclization of B4 to give B5.

The compounds of formula (I) may be prepared by cyclization of certainprotected argininals (as for example, A3 or B4) by means of treatingthem in a liquid mixture comprised of an acid and alcohol. Preferredmeans include treatment in a liquid mixture comprised of HCl andalcohol, p-toluenesulfonic acid and alcohol, pyridiniump-toluenesulfonate and alcohol, and camphorsulfonic acid and alcohol.Especially preferred means include treatment in a liquid mixturecomprised of concentrated HCl and an alcohol at 0°-30° C., morepreferably at 20°-25° C.

Where methyl is desired as R₂, more especially preferred means includetreatment in concentrated HCl in absolute methanol at 0°-30° C.,preferably at 20°-25° C. Where ethyl is desired as R₂, more especiallypreferred means include treatment in concentrated HCl in absoluteethanol at 0°-30° C., preferably at 20°-25° C. Where n-propyl is desiredas R₂, more especially preferred means include treatment in concentratedHCl in n-propanol at 0°-30° C., preferably at 20°-25° C. Where isopropylis desired as R₂, more especially preferred means include treatment withconcentrated HCl in isopropanol at 0°-30° C., preferably at 20°25° C.

3. Utility

The compounds and methods of the present invention are useful for makingpeptidyl argininals. Certain peptidyl argininals are useful as enzymeinhibitors or in vitro diagnostic reagents. For example, the peptidylargininals described in Example 11 (2-PrPen-Asp(OCH₃)-Pro-Arg-al) andExample 14 (2-PrPen-Asp-Pro-Arg-al) herein have been reported to bethrombin inhibitors, as well as, inhibitors of blood clotting. Vlasuk etal., WO 93/15756 (Aug. 19, 1993). Thus, certain peptidyl argininals(such as those of Examples 11 and 14) made by using the compounds andmethods of the present invention are useful as additives to solutions toinhibit the enzyme activity of thrombin.

The use of anticoagulants as in vitro diagnostic reagents is well known.For example, the use of stoppered test tubes containing anticoagulantsand having a vacuum therein as a means to draw blood obtained byvenipuncture into the tube is well known in the medical arts. Kasten, B.L., "Specimen Collection", Laboratory Test Handbook, 2nd Edition,Lexi-Comp Inc., Cleveland pp. 16-17 (Edits. Jacobs, D. S. et al. 1990).Such tubes contain clot-inhibiting additives (such as heparin salts,EDTA salts, citrate salts or oxalate salts), and are useful for theisolation of mammalian plasma from the blood. As potent inhibitors ofblood clotting, certain peptidyl argininals (such as those described inExamples 11 and 14 of this application) are also useful as in vitrodiagnostic reagents for addition into blood collection tubes.

4. Methods

In another aspect, the present invention is directed to methods ofmaking peptidyl argininals. FIG. 3 depicts the reaction scheme for apreferred method of the present invention. Exemplars of this reactionscheme are disclosed in Examples 9-11 and 21-23.

The preferred methods of the present invention include those comprisingthe steps of:

(a) preparing a first intermediate having the formula: ##STR15## whereinR₅ is selected from the group consisting of benzyloxycarbonyl,isonicotinyloxycarbonyl, 2-chlorobenzyloxycarbonyl,4-methoxybenzyloxycarbonyl, t-butoxycarbonyl, t-amyloxycarbonyl,isobornyloxycarbonyl, adamantyloxycarbonyl,2-(4-biphenyl)-2-propyloxycarbonyl, 9-fluorenylmethoxycarbonyl andmethylsulfonylethoxycarbonyl;

R₆ is selected from the group consisting of alkyl of 1 to about 12carbon atoms and aralkyl of about 7 to about 15 carbon atoms either ofwhich can be substituted with a hydroxy, and --CO--Y, wherein Y ishydroxy, alkoxy of 1 to about 12 carbon atoms, aralkoxy of about 7 toabout 15 carbon atoms, O-polymeric support or NH-polymeric support;

R7 is selected from the group consisting of hydrogen, Fmoc, nitro,benzyloxycarbonyl, t-butoxycarbonyl and adamantyloxycarbonyl; and

R₈ is selected from the group consisting of hydrogen, alkyl of 1 toabout 12 carbon atoms, aryl of about 6 to about 14 carbon atoms, andaralkyl of about 7 to about 15 carbon atoms;

(b) chemically removing the R₅ group from said first intermediate usinga R₅ removing reagent to give a second intermediate;

(c) chemically coupling to said second intermediate, a protected aminoacid, protected amino acid analog or protected peptide comprised ofabout 2 to about 30 amino acids, amino acid analogs, or a combination ofamino acids and amino acid analogs, using a coupling agent, to give athird intermediate having the formula: ##STR16## wherein X is aprotecting group,

k is an integer from 1 to 30, and

AA₁ -AA₂ . . . AA_(k) is an amino acid, amino acid analog or peptidecomprised of k amino acids, amino acid analogs or combination of aminoacids and amino acid analogs;

(d) chemically removing the R₇ group from said third intermediate, whenR₇ is not hydrogen, using a R₇ removing reagent to give a fourthintermediate having the formula: ##STR17## (e) chemically hydrolyzingsaid fourth intermediate with a hydrolyzing reagent which comprises anaqueous acid to chemically hydrolyze said fourth intermediate to give apeptidyl argininal.

As used herein, the term "peptidyl argininal" refers to a peptide inwhich the C-terminal amino acid is either L-argininal or D-argininal.The term "peptide" refers to to oligomers of amino acids which arelinked by peptide bonds. According to the conventional representation,the amino group at the N-terminus appears to the left and the carboxylgroup at the C-terminus to the right.

a. Preparing Intermediate

The preferred first intermediates (as shown above and as formula 1 inFIG. 3) used in the method of the present invention are prepared asdescribed above.

Preferred first intermediates include those wherein R₈ is hydrogen oralkyl of 1 to about 12 carbon atoms. Suitable alkyls for R₈ includemethyl, ethyl, 1-propyl, 2-methyl-1-propyl, 2,2-dimethyl-1-propyl,2-propyl, 2-methyl-2-propyl, 1-butyl, 2-butyl, 3-butyl,3-methyl-1-butyl, 1-pentyl, cyclopentyl, 1-hexyl, cyclopentylmethyl,cyclohexyl, cyclohexylmethyl, 1-heptyl, 4-heptyl, octyl, nonanyl,dodecanyl, adamantyl or adamantylmethyl. Especially preferred compoundsinclude those wherein R₈ is hydrogen, methyl, ethyl or propyl. Moreespecially preferred compounds include those wherein R₈ is hydrogen.

Preferred first intermediates include those wherein R₇ is Fmoc, nitro orbenzyloxycarbonyl. In this case, preferred compounds include thosewherein R₅ is t-butoxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl,adamantyloxycarbonyl, 4-methoxybenzyloxycarbony, or2-(4-biphenyl)-2-propyloxycarbonyl. Especially preferred firstintermediates include those wherein R₅ is t-butoxycarbonyl. Moreespecially preferred first intermediates include those wherein R₃ isnitro and R₁ is t-butoxycarbonyl.

Alternatively, preferred first intermediates include those wherein R₇ ist-butoxycarbonyl or adamantyloxycarbonyl. In this case, the preferredfirst intermediates include those wherein R₅ is benzyloxycarbonyl,isonicotinyloxycarbonyl, 2-chlorobenzyloxycarbonyl,9-fluorenylmethoxycarbonyl or methylsulfonylethoxycarbonyl. Especiallypreferred first intermediates include wherein R₅ is benzyloxycarbonyl.More especially preferred first intermediates include those wherein R₇is t-butoxycarbonyl and R.sub.≡ is benzyloxycarbonyl.

The preferred first intermediates include those wherein R₆ is alkyl of 1to about 12 carbon atoms optionally substituted with --CO--Y. Suitablealkyls for R₆ include methyl, ethyl, 1-propyl, 2-methyl-1-propyl,2,2-dimethyl-1-propyl, 2-propyl, 2-methyl-2-propyl, 1-butyl, 2-butyl,3-butyl, 3-methy-1-butyl, 1-pentyl, cyclopentyl, 1-hexyl,cyclopentylmethyl, cyclohexyl, cyclohexylmethyl, 1-heptyl, 4-heptyl,octyl, nonanyl, dodecanyl, adamantyl or adamantylmethyl. Especiallypreferred first intermediates include those wherein R₆ is methyl, ethyl,propyl or isopropyl. More especially preferred first intermediatesinclude those wherein R₆ is ethyl.

Preferred first intermediates of the present invention also includethose wherein R₇ is Fmoc and R₆ is --CO--Y, wherein Y is O-polymericsupport or NH-polymeric support. Preferred polymeric supports includeaminomethylated polystyrene resin, p-benzyloxybenzyl alcohol resin, Rinkamide resin, Merrifield resin and MBHA resin. In this case, thepreferred compounds include those wherein R₅ is hydrogen,t-butoxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl,adamantyloxycarbonyl, 4-methoxybenzyloxycarbonyl, or2-(4-biphenyl)-2-propyloxycarbonyl. More especially preferred compoundsinclude those wherein R₅ is hydrogen or t-butoxycarbonyl. Especiallypreferred compounds include those wherein R₇ is Fmoc and R₅ is hydrogenor t-butoxycarbonyl.

Certain especially preferred first intermediates include those havingthe formula: ##STR18## b. Chemically removing R₅ group

As depicted in FIG. 3, the R₅ group of the first intermediate 1 ischemically removed to give third intermediate 2.

Where R₅ is t-butoxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl,adamantyloxycarbonyl, 4-methoxybenzyloxycarbonyl or2-(4-biphenyl)-2-propyloxycarbonyl, the preferred means of chemicallyremoving the R₅ group from such first intermediates include theirtreatment with a liquid mixture comprised of an acid and solvent. Forexample, preferred means include chemically removing such R₅ group bytreatment with HCl in alcohol, trifluoroacetic acid in a chlorinatedhydrocarbon solvent, HCl in acetic acid, HCl in ethereal solvents, HClin ethyl acetate or methyl acetate, p-toluenesulfonic acid in toluene.Especially preferred means of chemically removing the R₅ group includetreatment with trifluoroacetic acid in dichloromethane at 0°-30° C.,more preferably at 20°25° C. Where R₆ is methyl, another more especiallypreferred means of chemically removing the R₅ group include treatmentwith HCl in absolute methanol at 0°-10° C., preferably at 0°-5° C. WhereR₆ is ethyl, another more especially preferred means of chemicallyremoving the R₅ group include treatment with HCl in absolute ethanol at0°-10° C., preferably at 0°-5° C. Where R₆ is n-propyl, another moreespecially preferred means of chemically removing the R₅ group includetreatment with HCl in n-propanol at 0°-10° C., preferably at 0°-5° C.Where R₆ is isopropyl, another more especially preferred means ofchemically removing the R₅ group include treatment with HCl inisopropanol at 0°10° C., preferably at 0°5° C.

Alternatively, where R₅ is benzyloxycarbonyl, isonicotinyloxycarbonyl or2-chlorobenzyloxycarbonyl, the preferred means of chemically removingthe R₅ group of such first intermediates include their treatment withhydrogen gas, or a source of hydrogen gas, in a liquid mixture comprisedof catalyst and solvent. For example, the preferred means of chemicallyremoving such R₅ groups include treatment with hydrogen gas and platinumor palladium in a liquid mixture comprised of alcohol, with1,4-cyclohexadiene and platinum or palladium in a liquid mixturecomprised of alcohol, or with ammonium formate and platinum or palladiumin a liquid mixture comprised of aqueous acetic acid. The more preferredmeans of chemically removing R₅ groups include their treatment withhydrogen gas and palladium in a liquid mixture comprised of alcohol andacid. Where R₆ is methyl, another more especially preferred means ofchemically removing R₅ groups include treatment with hydrogen gas and10% palladium on carbon in a liquid mixture comprised of methanol andHCl. Where R₆ is ethyl, another more especially preferred means ofchemically removing R₅ groups include treatment with hydrogen gas and10% palladium on carbon in a liquid mixture comprised of ethanol andHCl. Where R₆ is n-propyl, another more especially preferred means ofchemically removing R₅ groups include treatment with hydrogen gas and10% palladium on carbon in a liquid mixture comprised of n-propanol andHCl. Where R₆ is isopropyl, another more especially preferred means ofchemically removing R₅ groups include treatment hydrogen gas and 10%palladium on carbon in a liquid mixture comprised of isopropanol andHCl.

c. Chemically coupling

As depicted in FIG. 3, X-HN-AA₁ -AA₂ . . . AA_(k) -OH is coupled to 2 togive a third intermediate 3.

X-HN-AA₁ -AA₂ . . . AA_(k) -OH represents a protected amino acid,protected amino acid analog or protected peptide comprised of aminoacids, amino acid analogs or combination of amino acids and amino acidanalogs, wherein X-HN-AA₁ -AA₂ . . . AA_(k) -OH has a free C-terminalcarboxy group, and "k" is an integer, which is the number of aminoacids, amino acid analogs, or the combination of amino acids and aminoacid analogs which comprise X-HN-AA₁ -AA₂ . . . AA_(k) -OH. Where "k" is1, X-HN-AA₁ -OH is a protected amino acid or protected amino acidanalog. Where "k" is 2 to 20, X-HN-AA₁ -AA₂ . . . AA_(k) -OH is aprotected peptide comprised amino acids, amino acid analogs, or aminoacids and amino acid analogs, the total number of which equals "k".Preferred X-HN-AA₁ -AA₂ . . . AA_(k) -OH include those where "k" is 1 toabout 20. Especially preferred X-HN-AA₁ -AA₂ . . . AA_(k) -OH includethose wherein "k" is about 2 to about 10. More especially preferredX-HN-AA₁ -AA₂ . . . AA_(k) -OHs include those where "k" is about 2 toabout 5. "X" refers to a protecting group for the N-terminal amino acidor amino acid analog of X-HN-AA₁ -AA₂ . . . AA_(k) -OH. Preferredprotected amino acids for coupling include protected L-amino acids.

The preferred means of chemically coupling X-HN-AA₁ -AA₂ . . . AA_(k)-OH is coupled to 2 include formation of a peptide bond by usingconventional coupling reagents known in the art. See Bodanszky, N.,Peptide Chemistry, pp. 55-73, Springer-Verlag, New York (1988) andreferences cited therein. The chemical coupling may be either by meansof one-step or two-step coupling. In one-step coupling, X-HN-AA₁ -AA₂ .. . AA_(k) -OH is coupled directly to 2. Preferred coupling reagents forone-step coupling of the include DCC with HOBt, EDC with HOBt, HBTU,TBTU, HBTU with HOBt or TBTU with HOBt. In two-step coupling, anactivated ester or anhydride of the C-terminal carboxy group of X-HN-AA₁-AA₂ . . . AA_(k) -OH is formed prior to its coupling to 2.

X-HN-AA₁ -AA₂ . . . AA_(k) -OH is protected to prevent side reactionsand enhance correct coupling. Correct coupling requires that only theC-terminal carboxy group of X-HN-AA₁ -AA₂ . . . AA_(k) -OH is chemicallycoupled to the free amino group of 2. In particular, the N-terminalamino group, and if necessary, the side-chain groups of X-HN-AA₁ -AA₂ .. . AA_(k) -OH are protected by suitable protecting groups.

Where the synthesis of the desired peptidyl argininal is done bychemical coupling of a single X-HN-AA₁ -AA₂ . . . AA_(k) -OH having onits N-terminal amino group a non-removable protecting group as "X" maybe used. Suitable non-removable protecting groups include acyl groups orsulfonyl groups. Preferred non-removable protecting groups includeacetyl, 2-propylpentanoyl, 4-methylpentanoyl, t-butylacetyl,3-cyclohexylpropionyl, n-butanesulfonyl, benzylsulfonyl,4-methylbenzenesulfonyl, 2-naphthalenesulfonyl, 3-naphthalenesulfonyl,and 1-camphorsulfonyl.

Where the synthesis of the desired peptidyl argininal is done bystepwise addition of multiple X-HN-AA₁ -AA₂ . . . AA_(k) -OH,appropriate protecting groups as "X" and for the side chain functiongroups of the amino acids or amino acid analogs comprising X-HN-AA₁ -AA₂. . . AA_(k) -OH are selected which can be removed under non-adverseconditions. Non-adverse conditions means conditions of reaction orsynthesis which do not substantially adversely affect the skeleton ofthis peptide and/or its amino acid components.

Suitable N-terminal amino protecting groups which can be removed undernon-adverse conditions include: (a) aromatic urethane-type protectinggroups which include benzyloxycarbonyl, 2-chlorobenzyloxycarbonyl,9-fluorenylmethyloxycarbonyl, isonicotinyloxycarbonyl, and4-methoxybenzyloxycarbonyl; (b) aliphatic urethane-type protectinggroups which include t-butoxycarbonyl, t-amyloxycarbonyl,isopropyloxycarbonyl, 2-(4-biphenyl)-2-propyloxycarbonyl,allyloxycarbonyl and methylsulfonyl-ethoxycarbonyl; (c) cycloalkylurethane-type protecting groups which include adamantyloxycarbonyl,cyclopentyloxycarbonyl, cyclohexyloxycarbonyl and isobornyloxycarbonyl.Preferred N-terminal protecting groups include benzyloxycarbonyl andt-butoxycarbonyl. Especially preferred protecting groups includet-butoxycarbonyl.

Suitable side-chain protecting groups which can also be removed undernon-adverse conditions include: (a) for the side-chain amino grouppresent of lysine, protecting groups include any of the groups mentionedabove; (b) for the guanidino group of arginine, protecting groupsinclude nitro, benzyloxycarbonyl, t-butoxycarbonyl,2,2,5,7,8-pentamethyl-chroman-6-sulfonyl and2,3,6-trimethyl-4-methoxy-phenylsulfonyl; (c) for the hydroxyl group ofserine, threonine or tyrosine, protecting groups include t-butyl,benzyl, p-methoxybenzyl, p-nitrobenzyl, p-chlorobenzyl, o-chlorobenzyland 2,6-dichlorobenzyl; (d) for the carboxyl group of aspartic acid orglutamic acid, protecting groups include the methyl ester, ethyl ester,t-butyl ester and benzyl ester; (e) for the imidazole nitrogen ofhistidine, protecting groups include the benzyloxymethyl group; (f) forthe phenolic hydroxyl group of tyrosine, protecting groups includetetrahydropyranyl, t-butyl, trityl, benzyl, chlorobenzyl, 4-bromobenzyland 2,6-dichlorobenzyl; and (g) for the sulfhydryl group of cysteine,protecting groups include trityl, benzyl, 4-methoxybenzyl and2,4,6-trimethylbenzyl. Protecting groups for the N-terminal amino groupand side chain groups of amino acids and peptides such as thosedisclosed above are well known in the art. See Bodanszky, N., PeptideChemistry, pp. 74-103, Springer-Verlag, New York (1988) and referencescited therein.

It will be apparent to those skilled in the art that conditions forchemically removing protecting groups will vary. For example, certainprotecting groups such as triphenylmethyl and2-(4-biphenyl)-2-propyloxycarbonyl are very labile and can be cleavedunder mild acid conditions. Other protecting groups, such ast-butoxycarbonyl, t-amyloxycarbonyl, adamantyloxycarbonyl, and4-methoxybenzyloxycarbonyl are less labile and require moderately strongacids, such as trifluoroacetic, hydrochloric, or boron trifluoride inacetic acid, for their removal. Still other protecting groups, such asbenzyloxycarbonyl, 2-chlorobenzyloxycarbonyl, 4-nitrobenzyloxycarbonyland isopropyloxycarbonyl, are even less labile and require strongeracids, such as hydrogen fluoride, hydrogen bromide, borontrifluoroacetate in trifluoroacetic acid or trifluoromethanesulfonicacid in trifluoroacetic acid, for their removal.

In X-HN-AA₁ -AA₂ . . . AA_(k) -OH, the protecting groups (X) for theN-terminal amino group which are selected for use in the methods of thepresent invention can be chemically removed in the presence of theprotecting groups on the side-chain functional groups of the aminoacids, amino acid analogs, or combination of amino acids and amino acidanalogs comprising X-HN-AA₁ -AA₂ . . . AA_(k) -OH.

In selecting a particular N-terminal amino protecting group for use withcertain side-chain protecting groups in the synthesis of peptidylargininals, the following considerations may be determinative. AnN-terminal amino protecting group should: (a) render the N-terminalamino group inert under the conditions employed in the couplingreaction, (b) be readily removable after the coupling reaction underconditions that will not remove side-chain protecting groups nor alterthe structure of the peptide fragment, and (c) eliminate the possibilityof racemization upon activation immediately prior to coupling. Aside-chain protecting group should: (a) render the side-chain functionalgroup inert under the conditions employed in the coupling reaction, (b)be stable under the conditions employed in removing the N-terminal aminoprotecting group, and (c) should be readily removable upon completion ofthe synthesis of peptidyl argininal under reaction conditions that willnot alter its structure. The differential removal of a protecting groupin the presence of other protecting groups is also well known in theart. See Fauchere J-L and Schwyzer, "Differential Protection andSelective Protection in Peptide Synthesis", The Peptides, Volume 3, pp.203-252, Academic Press, New York (Edits. Gross, E. Meienhofer, J.1981).

The selection of a protecting group, X, for the N-terminal amino aminoacid or amino acid analog of X-HN-AA₁ -AA₂ . . . AA_(k) -OH depends onwhat protecting group, R₇, is employed in the first intermediate used.

Where R₇ is nitro or benzyloxycarbonyl group, the preferred groupsinclude t-butoxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl,adamantyloxycarbonyl, 4-methoxybenzyloxycarbony, or2-(4-biphenyl)-2-propyloxycarbonyl. Especially preferred protectinggroups include t-butoxycarbonyl. Alternatively, where R₇ ist-butoxycarbonyl or adamantyloxycarbonyl, the preferred protectinggroups include benzyloxycarbonyl, isonicotinyloxycarbonyl,2-chlorobenzyloxycarbonyl, 9-fluorenylmethoxycarbonyl ormethylsulfonylethoxycarbonyl. Especially preferred protecting groupsinclude benzyloxycarbonyl.

d. Chemically removing R₇ group

As depicted in FIG. 3, the R₇ group of the third intermediate 3 ischemically removed, when R₇ is not hydrogen, to give a thirdintermediate 4.

Where the R₇ group is nitro or benzyloxycarbonyl, preferred means tochemically remove it from a third intermediate include treating thethird intermediate with hydrogen gas in a liquid mixture comprised ofcatalyst, alcohol and acid. Especially preferred means includechemically removing such R₇ group by treatment with hydrogen gas onpalladium in a liquid mixture comprised of ethanol and acetic acid.

Where the R₇ group is t-butoxycarbonyl or adamantyloxycarbonyl,preferred means to chemically remove it from a third intermediateinclude treating a third intermediate with a liquid mixture comprised ofan acid and solvent. Especially preferred means include treatment withtrifluoroacetic acid in a chlorinated hydrocarbon solvent. Moreespecially preferred means include treatment with trifluoroacetic acidin dichloromethane at 0°-30° C., more preferably at 20°-25° C.

The R₇ group can also be removed using titanium trichloride in MeOH, Ch₂Cl₂, or DMF, according to the procedure of Freidinger, et al., J. OrgChem. 43:4800-4803 (1978).

e. Hydrolyzing

As depicted in FIG. 3, the fourth intermediate 4 is hydrolylzed to givethe peptidyl aldehyde 5. Preferred means of hydrolyzing a fourthintermediate include treatment with aqueous acid. Preferred aqueousacids include HCl, HPF₆, methanesulfonic acid, perchloric acid, sulfuricacid, trifluoroacetic acid, trifluoromethanesulfonic acid,toluenesulfonic acid. Where a fourth intermediate contains either a betaester of aspartic acid or gamma ester of glutamic acid, especiallypreferred acids include HPF₆. Where a fourth intermediate does notcontain a beta ester of aspartic acid or gamma ester of glutamic acid,especially preferred acids include HCl and HPF₆.

To assist in understanding the present invention, the following examplesfollow, which include the results of a series of experiments. Thefollowing examples relating to this invention are illustrative an shouldnot, of course, be construed as specifically limiting the invention.Moreover, such variations of the invention, now known or laterdeveloped, which would be within the purview of one skilled in the artare to be considered to fall within the scope of the present inventionhereinafter claimed.

EXAMPLES Example 1 Preparation of N-alpha-t-butoxycarbonyl-N^(g)-nitro-L-arginine lactam ##STR19##

N-alpha-t-butoxycarbonyl-N^(g) -nitroarginine (2.00 g, 6.3 mmole) wasdissolved in tetrahydrofuran (100 mL) by heating the solution to 50° C.The solution was allowed to cool to room temperature. N-methylpiperidine (0.84 mL, 6.9 mmole) was added, and the solution was cooledin an ice bath. Isobutylchloroformate (0.83 mL, 6.3 mmole) was added,and the reaction mixture was stirred at 0°° C. for 6 hours. The reactionmixture was stirred for 18 hours while the ice in the dewar was allowedto melt overnight. The solvent was removed under vacuum. The crudeproduct was dissolved in 20% ethyl acetate/dichloromethane (10 mL), andwas purified by flash chromatography through a 3×5 cm column of silicagel using 20% ethyl acetate/dichloromethane as eluent. 125 mL of eluentwas collected. The solvent was removed under vacuum to afford 1.39 g(74% crude yield) of the title compound as a white foam. R_(f) =0.44(silica gel, 5% isopropanol in dichloromethane). Isobutanol was presentas an impurity. This compound may be further purified byrecrystallization from dichloromethane/hexanes or ethanol/water.

Example 2 Preparation of N-alpha-t-butoxycarbonyl-N^(g)-nitro-L-argininal ##STR20## (a) Procedure 1

To a stirred solution of LiAlH₄ in tetrahydrofuran (3.8 mL of a 1.0Msolution, 3.8 mmole), cooled in an ice bath, was added dropwise ethylacetate (0.43 mL, 3.8 mmole) in tetrahydrofuran (5 mL). The solution wasstirred for 30 minutes at 0° C. to preform LiAlH₂ (OEt)₂.

The solution of this LiAlH₂ (OEt)₂ was added dropwise to a stirredsolution of compound of Example 1 (0.92 g, 3.1 mmole) in tetrahydrofuran(5 mL). After 30 minutes, the reaction is quenched with 1.0NHCl/tetrahydrofuran (2 mL of a 1:1 mixture). 1.0N HCl (20 mL) was added,and the solution was extracted three times with ethyl acetate (20 mLeach). The combined organic layers were washed with water (5 mL),saturated sodium bicarbonate (5 mL) and twice with brine(5 mL each),dried over anhydrous magnesium sulfate, filtered and the solvent wasremoved under vacuum to give 0.94 g (100% yield) of the title compoundas an off-white solid.

(b) Procedure 2

Alternatively, the title compound was made by the procedures whichfollow.

A 12 liter four-necked round bottom flask equipped with an overheadstirring apparatus was flame dried under a strong stream of nitrogen.After the flask had cooled, 120.0 g of N-alpha-t-butoxycarbonyl-N^(g)-nitro-L-arginine (376 mmole, 1 equivalent) was added under a blanket ofnitrogen followed by the addition of 6 liters of anhydroustetrahydrofuran (Aldrich sure-seal) via canula. The flask was thenfitted with a thermometer and the resulting suspension was warmed to 50°C. with a heat gun while stirring. The reaction mixture was cooled to 5°C. with an ice bath and further cooled to -5° C. with an ice/acetonebath.

During the time it took for this solution to reach -5° C., 36.66 g ofN-methyl-O-methylhydroxyamine hydrochloride (376 mmole, 1.0 equivalent)was weighed out in a 500 mL flask and suspended in 300 mL ofdichloromethane. This suspension was sparged with nitrogen for 5minutes, cooled to 0° C. and 46 mL of N-methylpiperidine (1.0equivalent) was added via syringe under nitrogen. The mixture wassonicated briefly to insure complete dissolution/free base formation andrecooled to 0° C. in an ice bath while still under nitrogen. Theresulting solution of free base was used later.

When the above arginine solution had reached -5° C., 45 mL ofN-methylpiperidine was added via syringe followed 5 minutes later by theaddition of 46 mL of isobutyl chloroformate (0.95 equivalent) viasyringe. The resulting solution was stirred for 15 minutes at -5° C.After this time, the free base solution of N-methyl-O-methylhydroxylamine generated above was added via canula over about 15minutes. Stirring was continued at -5° C. for another 1.5 hours at whichtime thin layer chromatography (silica gel, 1:10:90 aceticacid/methanol/dichloromethane) indicated that the reaction was complete.The reaction mixture was filtered while still cold, the salts washedwith 400 mL of cold tetrahydrofuran and the filtrate concentrated undervacuum on a rotary evaporator to yield a yellow foam.

The crude intermediate was taken up in 300 mL of dichloromethane andapplied to a column of silica gel (70-230 mesh, 7×50 cm). The column wasfirst eluted with 2 liters of dichloromethane followed by 2 liters of 2%methanol in dichloromethane. This was followed by elution with 5%methanol in dichloromethane until all of the product had been eluted(the eluant was checked for UV activity and five one-liter fractionswere collected once this UV activity was apparent). Fractions containingpure product were pooled and concentrated under vacuum and pumped onovernight to yield 120.1 g (88% yield) of N-alpha-t-butoxycarbonyl-N^(g)-nitro-L-arginine-(N-methyl, N-methoxyamide) as light yellow foam. Thisfoam was taken up in 300 mL of dichloromethane, 300 mL of toluene, andthe volatiles were once again removed under vacuum to remove anyresidual water or methanol.

120.1 g of N-alpha-t-butoxycarbonyl-N^(g) -nitro-L-arginine-(N-methyl,N-methoxyamide) (331.4 mmole) was taken up in 2.8 liters of dry (Aldrichsure-seal) tetrahydrofuran and transferred to a dry 5 liter 4-neckedround bottom flask equipped with a mechanical stirrer and a lowtemperature thermometer. The solution was cooled to -70° C. with a dryice/acetone bath and 300 mL of 1M LiAlH₄ in tetrahydrofuran was added bycanula transfer directly from 100 mL Aldrich sure-seal bottles. Anadditional 50 mL of 1M LiAlH₄ in tetrahydrofuran was added via syringe(total 331 mL). During the additions, the reaction temperature was keptbelow -60° C. The reaction was stirred for 0.5 hours at -70° C., thecooling bath removed, and the reaction was slowly allowed to warm to 0°C. (about 2.5 hours). Between -30° C. and -20° C. a thick slurryresulted. When the reaction mixture obtained 0° C., a small aliquot wasremoved and partitioned between ethyl acetate/2M potassium bisulfate.The organic layer analyzed by thin layer chromatography (silica gel,ethyl acetate).

When the reaction was judged to be complete, it was cooled to -70° C.and 503 mL of 2M potassium bisulfate was added via dropping funnel at aslow enough rate to keep the reaction temperature below -30° C. Thecooling bath was removed and the reaction mixture was allowed to come to0° C. over the course of 2 hours at which time a white precipitate wasfiltered off. The solids were washed with 500 mL of coldtetrahydrofuran. The filtrate was concentrated under vacuum on a rotaryevaporator until most of the tetrahydrofuran was removed and theremaining white sludge was mostly aqueous. The crude product was takenup in 1.5 liters of ethyl acetate and washed with 0.2M HCl (2×200 ml).The HCl extracts were back-extracted with 400 mL of ethyl acetate andthe organics were combined and extracted with saturated sodiumbicarbonate (2×200 mL). The bicarbonate extracts were alsoback-extracted with 400 ml of ethyl acetate. The organics were thencombined and washed with brine (200 mL) followed by drying overanhydrous sodium sulfate. The solution was filtered, concentrated undervacuum on a rotary evaporator and pumped on overnight to yield a whitesolid (89.0 g) of crude title compound. This was chromatographed onsilica gel and eluted with a gradient of 0 to 10% methanol indichloromethane. The pure fractions were combined and evaporated toyield the title compound as a white solid (75 g, 74%).

Example 3 Preparation of N-alpha-t-butoxycarbonyl-N^(g)-nitro-L-argininal ethyl cyclol ##STR21##

The compound of Example 2 (41.60 g, 0.137 mole) was dissolved in ethanol(200 mL) and concentrated HCl (1 mL) was added. After the reaction wascomplete by TLC (silica gel, 10% methanol in dichloromethane), thesolvent was removed under vacuum. The crude product was purified byflash chromatography through a column of silica gel (230-400 mesh) using0-10% ethyl acetate/dichloromethane as eluent. The combined fractionsyielded 36.88 g (81%) of the title compound as pale yellow foam. R_(f)=0.62 (silica gel, 5% methanol in dichloromethane).

Example 4 Preparation of N^(g) -nitro-L-argininal ethyl cyclol,trifluoroacetate salt ##STR22##

The compound of Example 3 (1.26 g) was treated with 50% trifluoroaceticacid/dichloromethane (10 mL) for 35 minutes. The solution was addeddropwise to diethyl ether (100 mL) while swirling. The resultingprecipitate was filtered and washed with diethyl ether. The light yellowpowder was dried under vacuum to yield (1.20 g, 91%) of the titlecompound.

Example 5 Preparation of N^(g) -nitro-L-argininal ethyl cyclol,hydrochloride salt ##STR23##

To a solution of the compound of Example 3 (35 g) in 500 mL of absoluteethanol at 0° C. was added slowly 500 mL of absolute ethanol saturatedwith HCl(g). This mixture was allowed to warm to 25° C. and checked bythin-layer chromatography. The appearance of a very polar product wasthe desired compound. Most of the HCl was removed with a stream of drynitrogen and the resulting organic solvent was removed under vacuum. Theresulting 33 g of the title compound as a yellow-white solid was usedwithout further purification.

Example 6 Preparation of Boc-L-aspartyl-beta-(methylester)-L-proline-O-benzyl ester ##STR24##

To a solution of isobutyl chloroformate (40.2 mL, 0.310 mole) and 1000mL of ethyl acetate at 0° C. was added slowly N-methylmorpholine (51.2mL, 0.465 mole). This mixture was stirred for 10 minutes with amechanical stirrer. Boc-L-aspartic acid-beta-methyl ester (75 g, 0.283mole) was added as a solid. The resulting solution was stirred for 15minutes. Next, solid L-proline-O-benzyl ester hydrochloride salt (75 g,0.310 mole) was added followed by the slow addition ofN-methylmorpholine (44.4 mL, 0.403 mole). After 30 minutes, the ice bathwas removed and the reaction was monitored by thin layer chromatography(silica gel, 5:95 methanol/dichloromethane). After about 2 hours, thereaction was completed, and the resulting organic phase was poured into1 liter of water. The organic phase was separated and washed three timeswith 300 mL of 1N HCl, one time with 300 mL saturated sodium bicarbonateand one time with 100 mL of brine. The organic phase was dried overanhydrous magnesium sulfate, filtered and the solvent was removed undervacuum. The yield of the yellow oil of the title compound was 120.2 g(91%). Rf=0.76 (silica gel, 5:95 methanol/dichloromethane).

Example 7 N-(2-propylpentanoyl)-L-aspartyl-beta-(methylester)-L-proline- O-benzyl ester ##STR25##

To a solution of the compound of Example 6 (112.6 g, 0.259 mole) and 400mL of ethyl acetate at 0° C. was added with stirring 700 mL of ethylacetate saturated with HCl (g). After about 1 hour, the reaction iscompleted by thin-layer chromatography (silica gel, 5:95methanol/dichloromethane). After removing the solvent under vacuum, theresulting solid was suspended in 500 mL of ethyl acetate to give asolution of L-aspartyl-beta-(methyl ester)-L-proline-O-benzyl esterhydrochloride salt.

To a solution of isobutyl chloroformate (28.6 mL, 0.220) and 300 mL ofethyl acetate at 0° C. was added slowly N-methylmorpholine (31.3 mL,0.285 mole). This mixture was stirred at 0° C. for 10 minutes and then2-propylpentanoic acid (34.5 mL, 0.220 mole) was added. The resultingsolution was stirred for 30 minutes and then added to the suspension ofL-aspartyl-beta-(methyl ester)-L-proline-O-benzyl ester hydrochloridesalt prepared above at 0° C. To this suspension was added slowlyN-methylmorpholine (31.3 mL, 0.389 mole). The ice bath was removed after30 minutes and the reaction mixture was allowed to warm to 25° C. Afterabout 3 hours, the reaction was complete as determined by thin-layerchromatography (silica gel, 5:95 methanol/dichloromethane) and theresulting organic phase was poured into 1 liter of water. The organicphase was separated and washed with thee times with 1N HCl (3×100 mL),three time with saturated sodium bicarbonate (3×100 mL) and one timewith brine (100 mL). The organic phase was dried over anhydrousmagnesium sulfate, filtered and the solvent was removed under vacuum togive a residue.

The residue was chromatographed on silica gel (230-400 mesh, 14×70 cmcolumn) and eluted with a gradient of 0 to 3% methanol indichloromethane. The solvents were evaporated to yield of the 106.8 g(90%) of the title compound as a yellow oil. Rf=0.73 (silica gel, 5:95methanol/dichloromethane).

Example 8 N-(2-propylpentanoyl)-L-aspartyl-beta-/methyl ester)-L-proline##STR26##

To a solution of the compound of Example 7 (111.6 g, 0.242 mole), 500 mLof methanol and 11 g of 10% palladium on carbon, wet withdichloromethane, was added hydrogen gas via a balloon. The reaction wasstirred overnight at 25° C. The following day, the reaction was completeas determined by thin-layer chromatography (silica gel, 5:95methanol/dichloromethane)). The solution was filtered through celite andand the celite was washed with dichloromethane (200 mL). The organicsolvent was evaporated under vacuum. The resulting white solid wastriturated with 300 mL of diethyl ether, filtered and dried to yield47.3 g (58%) the title compound. Rf=0.23 (silica gel, 20:80methanol/dichloromethane).

Example 9 Preparation of N-2propylpentanoyl)-L-aspartyl-beta-(methylester-L-prolyl-N^(g) nitro-L-argininal ethyl cyclol ##STR27##

To a stirred solution of the compound of Example 8(N-(2-propylpentanoyl)-L-aspartyl-beta-(methyl ester)-L-proline) (4.50g, 12 mmole), HBTU (4.61 g, 12 mmole), and HOBt (1.64 g, 12 mmol) inacetonitrile (70 mL) was added 4-methylmorpholine (5.30 mL, 48 mmole).After 10 minutes, the compound of Example 4 (N^(g) -nitroargininal ethylcyclol, trifluoroacetic acid salt) in acetonitrile (80 mL) was added.After 16 hours, the reaction mixture was concentrated, diluted withethyl acetate (500 mL) and water (300 mL). The organic layers werecombined and washed with 10% citric acid (3×300 mL), water (2×300 mL),saturated sodium bicarbonate (3×300 mL), and brine (2×100 mL). Thesolution was dried over anhydrous magnesium sulfate and the solvent wasremoved under vacuum. The residue was dissolved in ethyl acetate, andthe product precipitated. The solution was filtered and air dried togive the title compound (4.39 g, 62% yield) as a yellow powder.Analytical HPLC gave t_(R) =16.1 minutes (20-60% CH₃ CN/water containing0.1% trifluoroacetic acid, 25 mm Vydac C-18 column). Rf=0.85 (silicagel, 10% methanol in dichloromethane).

Example 10 Preparation of N-(2-propylpentanoyl)-L-aspartyl-beta-(methylester)-L-prolyl-L-argininal ethyl cyclol, acetate salt ##STR28##

The compound of Example 9 (4.39 g, 8 mmole), acetic acid (1.72 mL, 30mmole), and water (20 mL) in ethanol (75 mL) was hydrogenated over 10%palladium on carbon (0.44 g) for 72 hours at 45 psi. The reactionmixture was filtered through celite, washing with water. The solvent isremoved under vacuum to yield 5.2 g (100% yield) of the title compound.Analytical HPLC gave t_(R) =13.3 minutes (20--60% CH₃ CN/watercontaining 0.1% trifluoroacetic acid, 25 mm Vydac C-18 column.

Example 11 Preparation of N-(2-propylpentanoyl)-L-aspartyl-beta-(methylester)-L-prolyl-L-argininal, trifluoroacetate salt ##STR29##

The compound of Example 10 (10.0 g, 17 mmole) was dissolved in 50%aqueous acetonitrile (200 mL) and cooled in an ice bath. HPF₆ (60% byweight, 150 mL) was added slowly, and the cooling bath was removed.After 30 minutes, the reaction mixture was recooled in an ice bath, andquenched with aqueous sodium acetate (1.25 L of a 2.5M solution) to pH4, then filtered through a 2 micron filter. The filtrate was purifiedusing the Biotage HPLC, Vydac column #3, C-18, 4×60 cm column,acid/0-40% water in acetonitrile containing 0.1% trifluoroacetic acid.The fractions were analyzed for purity by analytical HPLC (20-60% CH₃CN/water containing 0.1% trifluoroacetic acid, 25 mm Vydac C-18 column),combined and the acetonitrile was removed under reduced pressure. Theremaining water was removed by lyophilization to give the title compound(4.26 g, 41% yield, 99% purity) as a white powder. 1.03 g (10%, 91%purity) was recovered from additional fractions. Fast atom bombardmentmass spectrometry confirmed the theoretical molecular weight of 510.

Example 12 Preparation ofN-(2-propylpentanoyl)-L-aspartyl-L-prolyl-N^(g) -nitro-L-argininal ethylcyclol ##STR30##

The compound of Example 9 (0.24 g, 0.41 mmole) was suspended in methanol(2 mL) and 1.0M LiOH (1.0 mL) was added dropwise. After 1 hour, thereaction mixture was diluted with water (10 mL) and washed with 2×3mLethyl acetate. The aqueous layer was adjusted with 1.0N HCl to pH 1.5,extracted with 3×5 mL ethyl acetate. The organic layers were combinedand washed with 2×mL brine, and dried over anhydrous magnesium sulfate.The solvent was reduced to approximately 10 mL and upon sitting a solidcrystallized out. The solid was filtered and air dried to give the titlecompound (110 mg, 47% yield) as an off-white crystals. Analytical HPLCgave t_(R) =13.3 minutes (20-60% CH₃ CN/water containing 0.1%trifluoroacetic acid, 25 mm Vydac C-18 column).

Example 13 Preparation ofN-(2-propylpentanoyl)-L-aspartyl-L-prolyl-L-argininal ethyl cyclol,acetate salt ##STR31##

The compound of Example 12 (1.4 g, 2.2 mmole) in ethanol/aceticacid/water (4:1:1, 150 mL) was hydrogenated over 10% palladium on carbon(0.70 g) for 22.5 hours at 40 psi. The solution was filtered throughcelite, then the celite was washed with water. The solvent was removedunder vacuum to yield 1.3 g (100% yield) of the title compound.Analytical HPLC gave t_(R) =11.2 minutes (20-60% CH₃ CN/water containing0.1% trifluoroacetic acid, 25 mm Vydac C-18 column).

Example 14 Preparation ofN-2-propylpentanoyl)-L-aspartyl-L-prolyl-L-argininal, trifluoroacetatesalt ##STR32##

The compound of Example 13 (0.400 g, 0.68 mmole) was dissolved in water(30 mL) and cooled in an ice bath. Concentrated HCl (12N, 10 mL) wasadded and the cooling bath is removed. After 3.25 hours, the reactionmixture was quenched with aqueous sodium acetate (2.5M, 10 mL), thenfiltered through a 2 micron filter. The filtrate was purified bypreparative HPLC (5×25 cm Vydac C-18 column, 0-40% acetonitrile/watercontaining 0.1% trifluoroacetic acid). Fractions were combined to give120 mg (29% yield) of the title compound. Fast atom bombardment massspectrometry confirmed the theoretical molecular weight of 496.

Example 15 Preparation of S-(t-butyl acetate)-L-cysteine ##STR33##

A 360 mL aqueous solution of commercially available (Aldrich) L-cysteinehydrochloride monohydrate (60.0 g, 341.7 mmole) and sodium hydroxide(27.33 g, 683.4 mmole), at room temperature, was treated with a solutionof t-butyl bromoacetate (72.3 g, 370.6 mmole) in 130 mL of dioxane over30 minutes. The reaction was stirred for 18 hours, during which time athick precipitate formed. The solid was filtered off, washed withdiethyl ether (100 mL) and dried under high vacuum at 40° C. to give82.5 g (103.8% crude yield includes occluded inorganic salt) of thetitle compound.

Example 16 Preparation of N-Boc-S-(t-butyl acetate)-L-cysteine ##STR34##

The compound of Example 15 (82.5 g, 341.7 mmole) and sodium bicarbonate(33.96 g, 404 mole) were suspended in 600 mL of deionized water. Asolution of di-t-butyl dicarbonate (80.88 g, 370 mole) in 350 mL ofdioxane was added and the slurry was stirred for 18 hours.

The slurry was extracted with diethyl ether (2×100 mL). The slurry waslayered with ethyl acetate (200 mL) and acidified with in hydrochloricacid to pH 2 (pH papers). The resulting organic layer was saved and theremaining aqueous layer was further extracted with ethyl acetate (2×200mL). The organic extracts were combined, washed with brine, dried withanhydrous magnesium sulfate and the solvent evaporated under vacuum toyield 84.3 g (74.6% yield) of the title compound as a clear oil. Thinlayer chromatography analysis of the title compound showed a single spotwith Rf=0.55, (silica; 90:10:2 dichloromethane/methanol/acetic acid).

Example 17 Preparation of N-Boc-S-(t-butylacetate)-L-cysteine-L-proline-O-benzyl ester ##STR35##

The compound of Example 16 (31.89 g, 95.06 mmole) and L-proline-O-benzylester hydrochloride (22.98 g, 95.06 mmole) were suspended in 140 mL ofacetonitrile and 120 mL of dimethylformamide at 0° C., then BOP (42.0 g,95.06 mmole) and N-methylmorpholine (28.84 g, 285.18 mmole) were added.The ice bath was removed after 30 minutes and the reaction was stirredfor 18 hours at room temperature. The reaction mixture was reduced involume under vacuum at 25° C. to give an oil. The oil was dissolved inethyl acetate (250 mL), then successively washed with in hydrochloricacid (1×50 mL), saturated sodium bicarbonate (1×50 mL) and brine (1×50mL). The organic layer was dried with anhydrous magnesium sulfate andevaporated under vacuum to give crude product.

The crude produce was purified by column chromatography on silica gel,eluting with 55:45 hexane/ethyl acetate to yield 27.91 g (57.9% yield)of the title compound as an oil. Thin layer chromatography analysis ofthe title compound showed a single spot with Rf=0.65 (silica, 3:2 ethylacetate/hexane).

Example 18 Preparation of N-Boc-S-(t butyl acetate)-L-cysteinesulfone-L-proline-O-benzyl ester ##STR36##

The compound of Example 17 (27.9 g, 55.07 mmole) was dissolved in 300 mLof glacial acetic acid, sodium perborate tetrahydrate (42.36 g, 275.35mole) was added and the mixture was heated to 55° C. After 2.5 hours atthis temperature, the reaction mixture was diluted with 1 liter ofbrine, the aqueous layer was extracted with ethyl acetate (4×250 mL) andthe combined organic extracts were dried with anhydrous magnesiumsulfate. This solution was filtered and evaporated under vacuum, thenrepeatedly azeotroped with toluene (200 mL) under vacuum to removeacetic acid. The residual slurry was dissolved in ethyl acetate (200mL), filtered and the filtrate evaporated to yield 29.7 g (100% yield)of the title compound as a white solid. Thin layer chromatographyanalysis of the title compound showed a single spot with Rf=0.60(silica, 3:2 ethyl acetate/hexane).

Example 19 Preparation of N-(2-propylpentanoyl)-S-(t-butylacetate)-L-cysteine sulfone-L-proline-O-benzyl ester ##STR37##

A solution of the compound of Example 18 (5.0 g, 9.28 mmole) in 105 mLof sieve-dried ethyl acetate was prepared. To this, 26 mL of 5.7Nanhydrous hydrochloric acid/ethyl acetate (that had been generated insitu from acetyl chloride and methanol) was added. This mixture wasstirred for several hours at room temperature until all startingmaterial was consumed. The mixture was evaporated under vacuum and theresulting oil was dissolved in acetonitrile and then evaporated undervacuum. This was done three times.

The remaining oil was suspended in 35 mL of acetonitrile, cooled to icebath temperature, then 2-propylpentanoic acid (1.60 g, 11.4 mmole), BOP(4.10 g, 9.28 mmole) and N-methylmorpholine (3.75 g, 37.1 mmole) wereadded. The reaction was removed from the ice bath after 30 minutes andallowed to stir at room temperature for 18 hours. The reaction mixturewas reduced in volume under vacuum to an oil. The oil was taken up in200 mL ethyl acetate and washed successively with in hydrochloric acid(1×50 mL), saturated sodium bicarbonate (1×50 mL) and brine (1×50 mL).After drying with anhydrous magnesium sulfate, the organic layer wasevaporated under vacuum to give crude product.

The crude product was purified by column chromatography on silica gel,eluting with 3:2 hexane/ethyl acetate to yield 1.81 g (34.5% yield) ofthe title compound as a solid. Thin layer chromatography analysis of thetitle compound showed a single spot with Rf=0.50 (silica, 3:2 ethylacetate/hexane).

Example 20 Preparation of N-(2-propylpentanoyl)-S-(t-butylacetate)-L-cysteine sulfone-L-proline ##STR38##

The compound of Example 19 (1.81 g, 3.2 mmole) was dissolved intetrahydrofuran (50 mL), 0.5 g of 10% palladium on carbon was added andthe mixture was stirred under hydrogen gas at atmospheric pressure for18 hours.

After the catalyst was filtered off the reaction mixture, the solventwas removed under vacuum and the resulting oil was taken up in asolution of saturated sodium bicarbonate. This solution was thenextracted with ethyl acetate (1×150 mL) and the organic layer wasdecanted off. The remaining aqueous layer was layered with 100 mL ofethyl acetate and acidified with in hydrochloric acid to pH 2 (pHpapers). After the phases separated, the organic layer was saved and theaqueous layer was then further extracted with ethyl acetate (3×100 mL).

The organic extracts were combined and washed with brine, dried withanhydrous magnesium sulfate, filtered and evaporated under vacuum togive 1.30 g (yield 85.6%) of the title compound as a foamy solid. Thinlayer chromatography analysis of the title compound showed a single spotwith Rf=0.35 (silica, 90:10:2 dichloromethane/methanol/acetic acid).

Example 21 Preparation ofN-(2-propylpentanoyl)-S-(t-butylacetate)-L-cysteinesulfone-L-proline-N^(g) -nitro-L-argininal ethyl cyclol ##STR39##

The compound of Example 5 (N^(g) -nitro-L-argininal ethyl cyclol,hydrochloride salt) (0.70 g, 2.6 mmole) was dissolved with stirring in 6mL of dry dimethylformamide and 13 mL of dry acetonitrile. To thismixture was added N-methylmorpholine (1.4 mL, 13.1 mmole) followed bythe compound of Example 20 (0.96 g, 1.9 mmole) HOBt (0.53 g, 3.9 mmole)and HBTU (1.5 g, 3.9 mmole). After 16 hours, the reaction mixture wasdiluted with 600 mL ethyl acetate and extracted with 150 mL each ofwater, 1M aqueous HCl, water, saturated aqueous sodium bicarbonate andbrine. The organic phase was dried over anhydrous magnesium sulfate,filtered and the solvent removed under vacuum to give 1.3 g (95% yield)of the title compound as an off-white foam. Rf=0.50 (1:9methanol/dichloromethane).

Example 22 Preparation ofN-(2-propylpentanoyl)-S-(t-butylacetate)-L-cysteinesulfone-L-proline-N^(g) -nitro-L-argininal ethyl cyclol, acetate salt##STR40##

1.0 g of 10% palladium on carbon was placed in a 500 mL Parr bottlefollowed by 10 mL of water and 3.7 mL of glacial acetic acid was added.To this mixture was added, a solution of the compound of Example 21 (1.3g, 1.85 mmole) in 100 mL of methanol. The mixture was then shaken undera hydrogen atmosphere at 40 psi for 3 days. The catalyst was thenremoved by filtration and the filtrate concentrated under vacuum to givean oil. The residue was purified by preparative HPLC (5×25 cm Vydac C-18column, 10-40% acetonitrile/water containing 0.1% trifluoroacetic acid).Fractions were combined to give 0.7 g (58%) of the title compound.

Example 23 Preparation ofN-(2-Propylpentanoyl)-S-(carboxymethyl)-L-cysteinesulfone-L-proline-N^(g) -nitro-L-argininal, trifluoroacetate salt##STR41##

The compound of Example 22 (0.7 g, 1.1 mmole) was dissolved in 30 mL of50:50 water/acetonitrile with stirring and cooled to 0° C. in an icewater bath. To this solution was slowly added 40 mL of a 60 wt %solution of HPF₆ in water. After 1 hour, analytical HPLC (20-60% CH₃CN/water containing 0.1% trifluoroacetic acid, 25 mm Vydac C-18 column)showed the reaction to be complete and pH of the reaction mixture wasdecreased to about pH=4 using 2.5M aqueous sodium acetate. This mixturewas filtered through a plug of celite and then was purified bypreparative HPLC (1"×S" C18, 22 mL/min, 12-30% acetonitrile/watercontaining 0.1% trifluoroacetic acid) to give the title compound uponlyophilization of the pooled fractions. Fast atom bombardment massspectrometry confirmed the theoretical molecular weight of 574.

Example 24 Preparation of N-alpha-t-butoxycarbonyl-N^(g)-carbobenzyloxy-L-arginine ##STR42##

Alpha-N-tert-butyloxycarbonyl-L-arginine hydrochloride hydrate (82.1 g,250 mmoles) is dissolved in 5.0N sodium hydroxide (240 mL) and cooled to-5° C. to 0° C. Under vigorous stirring benzyl chloroformate (143 mL, 1mole) is added dropwise over a 55-minute period, while 5.0N sodiumhydroxide (250 mL) is added to the solution at such a rate that the pHof the mixture is maintained at 13.2-13.5. Stirring is continued at 0°C. for 1 hour after addition of the chloroformate is complete. Thereaction mixture is diluted with water (100 mL) and diethyl ether (500mL). The aqueous layer is separated and extracted with diethyl ether(2×40 mL). The aqueous layer is acidified with 3N sulfuric acid to pH3.0 (about 560 mL) and extracted with ethyl acetate (550 mL). Theaqueous layer is extracted with ethyl acetate (250 mL). The combinedethyl acetate extracts are washed with water, dried over anhydrousmagnesium sulfate and evaporated under reduced pressure. Theprecipitated product is triturated with diethyl ether, filtered, washedwith diethyl ether and air-dried to give the title compound.

Example 25 Preparation of N-alpha-t-butoxycarbonyl-N^(g)-carbobenzyloxy-L-arginine lactam ##STR43##

The compound from Example 24 (66.0 g, 162 mmole) is dissolved intetrahydrofuran (230 mL) and the mixture is cooled to -10° C. in anice-acetone bath. To the cold solution is added N-methylmorpholine (18.7mL, 170 mmoles) followed by isobutyl chloroformate (22.5 mL, 170mmoles). After stirring for 5 minutes, triethylamine (23.5 mL, 170mmoles) is added. The reaction mixture is stirred for an additional hourat -10° C., then for one hour at room temperature. The reaction mixtureis poured into ice water (1 L). The precipitated material is filtered,washed with cold water, dried under vacuum, and crystallized from ethylacetate to give the title compound.

Example 26 Preparation of N-alpha-t-butoxycarbonyl-N^(g)-carbobenzyloxy-L-argininal ##STR44##

To a stirred solution of LiAlH₄ in tetrahydrofuran (4.8 mL of a 1.0Msolution, 4.8 mmole), cooled in an ice bath, is added dropwise ethylacetate (0.54 mL, 4.8 mmole) in tetrahydrofuran (5 mL). The solution isstirred for 30 minutes at 0° C. to preform LiAlH₂ (OEt)₂.

The solution of this LiAlH₂ (OEt)₂ is added dropwise to a stirredsolution of the compound of Example 25 (1.50 g, 3.8 mmole) intetrahydrofuran (10 mL). After 30 minutes, the reaction is quenched with1.0N HCl/tetrahydrofuran (2.5 mL of a 1:1 mixture). 1.0N HCl (20 mL) isadded, and the solution is extracted three times with ethyl acetate(3×20 mL). The combined organic layers are washed with water (5 mL),saturated sodium bicarbonate (5 mL) and twice with brine (2×5 mL). Theextracts are dried over anhydrous magnesium sulfate, filtered and thesolvent is removed under vacuum to give the title compound.

Example 27 Preparation of N-alpha-t-butoxycarbonyl-N^(g)-carbobenzyloxy-L-argininal ethyl cyclol ##STR45##

The compound of Example 26 (1.00 g, 2.5 mmole) is dissolved in ethanol(10 mL) and concentrated HCl (0.05 mL) is added. After the reaction iscomplete, the solvent is removed under vacuum. The crude product may bepurified by flash chromatography through a column of silica gel (230-400mesh). Removal of the solvents under vacuum from the combined fractionswill yield the title compound.

Example 28 Preparation of N^(g) -carbobenzyloxy-L-argininal ethylcyclol, trifluoroacetate salt ##STR46##

The compound of Example 27 (0.80 g) is treated with 50% trifluoroaceticacid/dichloromethane (8 mL) for 30 minutes. The solution is diluted withtoluene, and the solvent is reduced under vacuum. The residue is againdiluted with toluene, and the solvent is removed under vacuum to givethe title compound. The resulting title compound is used without furtherpurification.

Example 29 Preparation of N^(g) -carbobenzyloxy-L-argininal ethylcyclol, hydrochloride salt ##STR47##

To a solution of the compound of Example 28 (3.5 g) in 50 mL of absoluteethanol at 0° C. is added slowly 50 mL of absolute ethanol saturatedwith HCl (g). This mixture is allowed to warm to 25° C. and checked bythin-layer chromatography. The HCl is removed with a stream of drynitrogen and the remaining organic solvent is removed under vacuum togive the title compound. The title compound is used without furtherpurification.

Example 30 Preparation of N-(2-propylpentanoyl)-L-aspartyl-beta-(methylester)-L-prolyl-N^(g) -Carbobenzyloxy-L-argininal ethyl cyclol ##STR48##

To a stirred solution of the compound of Example 8 (4.50 g, 12 mmole),HBTU (4.61 g, 12 mmole), and HOBt (1.64 g, 12 mmol) in acetonitrile (70mL) is added 4-methylmorpholine (5.30 mL, 48 mmole). After 10 minutes,the compound of Example 29 (5.2 g, 12 mmole) in acetonitrile (80 mL) isadded. After 16 hours, the reaction mixture is concentrated, dilutedwith ethyl acetate (500 mL) and water (300 mL). The organic layers arecombined and washed three times with 10% citric acid (3×300 mL), twotimes with water (2×300 mL), three times with saturated sodiumbicarbonate (3×300 mL), and two times with brine (2×100 mL). Thesolution is dried over anhydrous magnesium sulfate and the solvent isremoved under vacuum. The crude product may be purified by flashchromatography through a column of silica gel (230-400 mesh). Uponremoval of the solvents under vacuum the combined fractions will yieldthe title compound.

Example 31 Preparation of N-(2-propylpentanoyl)-L-aspartyl-beta-(methylester)-L-prolyl-L-argininal ethyl cyclol, acetate salt ##STR49##

The compound of Example 30 (4.39 g, 8 mmole), acetic acid (1.72 mL, 30mmole), and water (20 mL) in ethanol (75 mL) is hydrogenated over 10%palladium on carbon (0.44 g) for 5 hours at one atmosphere of hydrogen.The reaction mixture is filtered through celite, and the celite iswashed with water. The solvent is removed under vacuum to yield thetitle compound.

Example 32 Preparation of N-(2-propylpentanoyl-L-aspartyl-beta-(methylester)-L-prolyl-L-argininal, trifluoroacetate salt ##STR50##

The compound of Example 31 (10.0 g, 17 mole) is dissolved in 50% aqueousacetonitrile (200 mL) and cooled in an ice bath. HPF₆ (60% by weight,150 mL) is added slowly, and the cooling bath is removed. After 30minutes, the reaction mixture is recooled in an ice bath, and quenchedwith aqueous sodium acetate (1.25 L of a 2.5M solution) to pH 4, then isfiltered through a 2 micron filter. The filtrate is purified using theBiotage HPLC, Vydac column #3, C-18, 4×60 cm column, acid/0-40% water inacetonitrile containing 0.1% trifluoroacetic acid. The fractions areanalyzed for purity by analytical HPLC (20-60% acetonitrile-watercontaining 0.1% trifluoroacetic acid, 25 mm Vydac C-18 column), combinedand the acetonitrile is removed under reduced pressure. The remainingwater is removed by lyophilization to give the title compound.

Example 33 Preparation of N-alpha-t-butoxycarbonyl-N^(g)-nitro-L-argininal (carboethoxymethyl) cyclol ##STR51##

To a suspension of the compound of Example 2 (4.99 g, 16 mmole) andethyl glycolate (2.0 mL, 21 mmole) in dichloromethane (20 mL) was addedcamphorsulfonic acid (0.20 g, 1 mmole). After 24 hours, the reactionmixture was diluted with ethyl acetate (100 mL), washed with saturatedsodium bicarbonate and brine, dried over magnesium sulfate, and thesolvent was removed. The residue was chromatographed through flashsilica gel using 2% isopropanol/dichloromethane. The fractions werecombined to give a total of 2.59 g of the title compound in 40% yield.R_(f) =0.39 (silica gel, 10% isopropanol in chloroform).

Example 34 Preparation of N-alpha-t-butoxycarbonyl-N^(g)-nitro-L-argininal (carboxymethyl) cyclol ##STR52##

The compound of Example 33 (2.00 g, 5.1 mmol) is suspended in methanol(2 mL), cooled in an ice bath, and 1.0M lithium hydroxide (10.2 ml, 10.2mmole) is added dropwise. The ice bath is removed. After 1-16 hours, thesolution is diluted with water (100 mL) and washed with ethyl acetate(2×30 mL). The aqueous layer is acidified with 1.0N HCl to pH 1.5, thenextracted with ethyl acetate (3×50 ml). The combined organic layers arewashed twice with brine (1 ml each wash), and dried over magnesiumsulfate. Solvent is removed and the title compound is isolated.

Example 35 Preparation of N-alpha-t-butoxycarbonyl-N^(g)-nitro-L-argininal carbo(Merrifield resin)oxymethyl!cyclol ##STR53##

Merrifield resin (0.78 mmole/g substitution, 1.0 g, 0.78 mmole) issuspended in dimethylformamide (20 mL), and potassium fluoride (181 mg,3.1 mmole) and the compound of Example 34 (0.56 g, 1.6 mmole) are added.The reaction mixture is heated in an 80° C. oil bath while stirring for24 h, then allowed to cool to room temperature. The substitution of theresin is determined using a small sample of the substituted resin andthe picric acid assay (John M. Stewart, Janis D. Young, Solid PhasePeptide Synthesis, Pierce Chem Co. (1984) p. 107). The resin isfiltered, washed with dichloromethane (3×), dimethylformamide (4×),water (3×) and methanol (3×), and dried under vacuum to give the titlecompound.

Example 36 Preparation of N-alpha-t-butoxycarbonyl-N^(g)-nitro-L-argininal carbo(MBHA resin)oxy-methyl!cyclol ##STR54##

The compound of Example 35 (8.1 g, 22.4 mmole) is coupled to MBHA resin(1.12 mmole/g substitution, 20.0 g, 22.4 mmole) with BOP reagent (9.91g, 22.4 mmole) and 4-methylmorpholine (2.26 mL, 22.4 mmole) using thestandard protocol. (John M. Stewart, Janis D. Young, Solid Phase PeptideSynthesis, Pierce Chem Co. (1984) p. 73). After 24 hours, the resin iswashed with dichloromethane (3×), dimethylformamide (4×), trituratedwith 10% diisopropylethylamine in dimethylformamide (10 minutes forfirst wash, 20 minutes for second wash), then washed withdimethylformamide (4×). The efficiency of coupling is determined using asmall amount of the coupled resin and the ninhydrin test. The resin isdouble coupled with the compound of Example 35 (8.1 g, 22.4 mmole), BOPreagent (9.91 g, 22.4 mmole) and 4-methylmorpholine (2.26 mL, 22.4mmole) using the same protocol as before. After 24 hours, the resin iswashed with dichloromethane (3×), dimethylformamide (4×), and methanol(4×). The efficiency of coupling is determined using a small amount ofthe coupled resin and the ninhydrin test. The resin is dried undervacuum to give the title compound.

We claim:
 1. A compound of the formula: ##STR55## R₁ is selected fromthe group consisting of hydrogen, benzyloxycarbonyl,isonicotinyloxycarbonyl, 2-chlorobenzyloxycarbonyl,4-methoxybenzyloxycarbonyl, t-butoxycarbonyl, t-amyloxycarbonyl,isobornyloxycarbonyl, adamantyloxycarbonyl,2-(4-biphenyl)-2-propyloxycarbonyl, 9-fluorenylmethoxycarbonyl andmethylsulfonylethoxycarbonyl;R₂ is selected from the group consisting ofalkyl of 1 to about 12 carbon atoms and aralkyl of about 7 to about 15carbon atoms, either of which is optionally substituted with hydroxy,or, --C(O)--Y, wherein Y is hydroxy, alkoxy of 1 to about 12 carbonatoms, aralkoxy of about 7 to about 15 carbon atoms, O-polymeric supportor NH-polymeric support; R₃ is selected from the group consisting ofhydrogen, Fmoc, nitro, benzyloxycarbonyl, t-butoxycarbonyl andadamantyloxycarbonyl; and R₄ is selected from the group consisting ofhydrogen, alkyl of 1 to about 12 carbon atoms, aryl of about 6 to about14 carbon atoms and aralkyl of about 7 to about 15 carbon atoms; andsalts thereof.
 2. A compound of claim 1, wherein R₄ is hydrogen, methyl,ethyl or propyl.
 3. A compound of claim 2, wherein R₄ is hydrogen.
 4. Acompound of claim 3, where R₃ is nitro.
 5. A compound of claim 4,wherein R₂ is alkyl of 1 to about 12 carbon atoms.
 6. A compound ofclaim 5, wherein R₂ is methyl, ethyl, propyl or isopropyl.
 7. A compoundof claim 6, wherein R₂ is ethyl.
 8. A compound of claim 7, wherein R₁ ishydrogen, 4-methoxybenzyloxycarbonyl, t-butoxycarbonyl,t-amyloxycarbonyl, isobornyloxycarbonyl, adamantyloxycarbonyl or2-(4-biphenyl)-2-propyloxycarbonyl.
 9. A compound of claim 8, wherein R₁is hydrogen.
 10. A compound of claim 8, wherein R₁ is t-butoxycarbonyl.11. A compound of claim 3, where R₃ is benzyloxycarbonyl.
 12. A compoundof claim 11, wherein R₂ is alkyl of 1 to about 12 carbon atoms.
 13. Acompound of claim 12, wherein R₂ is methyl, ethyl, propyl or isopropyl.14. A compound of claim 13, where R₂ is ethyl.
 15. A compound of claim14, wherein R₁ is hydrogen, 4-methoxybenzyloxycarbonyl,t-butoxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl,adamantyloxycarbonyl or 2-(4-biphenyl)-2-propyloxycarbonyl.
 16. Acompound of claim 15, wherein R₁ is hydrogen.
 17. A compound of claim15, wherein R₁ is t-butoxycarbonyl.
 18. A compound of claim 3, where R₃is t-butoxycarbonyl.
 19. A compound of claim 18, wherein R₂ is alkyl of1 to about 12 carbon atoms.
 20. A compound of claim 19, wherein R₂ ismethyl, ethyl, propyl or isopropyl.
 21. A compound of claim 20, where R₂is ethyl.
 22. A compound of claim 21, wherein R₁ is hydrogen,benzyloxycarbonyl, isonicotinyloxycarbonyl or 2-chlorobenzyloxycarbonyl.23. A compound of claim 22, wherein R₁ is hydrogen.
 24. A compound ofclaim 22, wherein R₁ is benzyloxycarbonyl.
 25. A compound of claim 3,wherein R₃ is Fmoc.
 26. A compound of claim 25, wherein R₂ issubstituted with --C(O)--Y, wherein Y is O-polymeric support orNH-polymeric support.
 27. A compound of claim 26, wherein the polymericsupport is selected from the group consisting of aminomethylatedpolystyrene resin, p-benzyloxybenzyl alcohol resin, Merrifield resin,Rink amide resin, and MBHA resin.
 28. A compound of claim 27, whereinthe polymeric support is Merrifield resin or MBHA resin.
 29. A compoundof claim 26, wherein R₁ is hydrogen, 4-methoxybenzyloxycarbonyl,t-butoxycarbonyl, t-amyloxycarbonyl,isobornyloxycarbonyl,adamantyloxycarbonyl or2-(4-biphenyl)-2-propyloxycarbonyl.
 30. A compound of claim 29, whereinR₁ is hydrogen.
 31. A compound of claim 29, wherein R₁ ist-butoxycarbonyl.
 32. A compound of claim 1 selected from the groupconsisting of: ##STR56##
 33. A method of making a peptidyl argininalcomprising the steps of:(a) reacting a first intermediate having theformula: ##STR57## wherein R₅ is selected from the group consisting ofbenzyloxycarbonyl, isonicotinyloxycarbonyl, 2-chlorobenzyloxycarbonyl,4-methoxybenzyloxycarbonyl, t-butoxycarbonyl, t-amyloxycarbonyl,isobornyloxycarbonyl, adamantyloxycarbonyl,2-(4-biphenyl)-2-propyloxycarbonyl, 9-fluorenylmethoxycarbonyl andmethylsulfonylethoxycarbonyl;R₆ is selected from the group consisting ofalkyl of 1 to about 12 carbon atoms and aralkyl of about 7 to about 15carbon atoms, either of which is optionally substituted with hydroxy, or--C(O)--Y, wherein Y is hydroxy, alkoxy of 1 to about 12 carbon atoms,aralkoxy of about 7 to about 15 carbon atoms, O-polymeric support orNH-polymeric support; R₇ is selected from the group consisting ofhydrogen, Fmoc, nitro, benzyloxycarbonyl, t-butoxycarbonyl andadamantyloxycarbonyl; and R₈ is selected from the group consisting ofhydrogen, alkyl of 1 to about 12 carbon atoms, aryl of about 6 to about14 carbon atoms, and aralkyl of about 7 to about 15 carbon atoms; with aR₅ removing reagent which chemically removes the R₅ group from saidfirst intermediate to give a second intermediate of the formula:##STR58## (b) chemically coupling to the second intermediate of step(a), a protected amino acid, a protected amino acid analog or aprotected peptide of about 2 to about 30 amino acids, amino acidanalogs, or a combination of amino acids and amino acid analogs, using acoupling reagent to give a third intermediate having the formula:##STR59## wherein X is a protecting group, k is an integer from 1 to 30,and AA₁ -AA₂ . . . AA_(k) is an amino acid, amino acid analog or peptidecomprised of k amino acids, amino acid analogs or combination of aminoacids and amino acid analogs; (c) reacting the third intermediate with aR₇ removing reagent, when R₇ is not hydrogen, which chemically removesthe R₇ group to give a fourth intermediate having the formula: ##STR60##(d) reacting the fourth intermediate with a hydrolyzing reagent whichcomprises an aqueous acid to chemically hydrolyze said fourthintermediate to give said peptidyl argininal.
 34. A method of claim 33,wherein R₈ is hydrogen, methyl, ethyl or propyl.
 35. A method of claim34, wherein R₈ is hydrogen.
 36. A method of claim 35, wherein R₇ isnitro.
 37. A method of claim 36, wherein R₆ is alkyl of 1 about 12carbon atoms.
 38. A method of claim 37, wherein R₆ is methyl, ethyl,propyl or isopropyl.
 39. A method of claim 38, wherein R₆ is ethyl. 40.A method of claim 39, wherein R₅ is 4-methoxybenzyloxycarbonyl,t-butoxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl,adamantyloxycarbonyl and 2-(4-biphenyl)-2-propyloxycarbonyl.
 41. Amethod of claim 40, wherein said R₅ removing reagent is a liquid mixturecomprised of an acid and solvent.
 42. A method of claim 41, wherein saidacid is selected from the group consisting of HCl, trifluoroacetic acidand p-toluenesulfonic acid.
 43. A method of claim 42, wherein said acidis HCl and said solvent is ethanol.
 44. A method of claim 41, whereinsaid second intermediate is coupled to said X-AA₁ -AA₂ . . . AA_(k) -OHusing a coupling reagent selected from the group consisting of DCC withHOBt, EDC with HOBt, HBTU, TBTU, HBTU with HOBt, and TBTU with HOBt. 45.A method of claim 44, wherein R₇ is chemically removed from said thirdintermediate by treating with hydrogen gas in a liquid mixture comprisedof catalyst, alcohol and acid.
 46. A method of claim 45, wherein saidcatalyst is palladium.
 47. A method of claim 46, wherein said alcohol isethanol and said acid is acetic acid.
 48. A method of claim 47, whereinsaid hydrolyzing reagent is an aqueous acid selected from the groupconsisting of HCl, HPF₆, methane sulfonic acid, perchloric acid,sulfuric acid, trifluoroacetic acid, trifluoromethane sulfonic acid andtoluene sulfonic acid.
 49. A method of claim 48, wherein said aqueousacid is HPF₆ or HCl.
 50. A method of claim 49, wherein X is selectedfrom a group consisting of acetyl, 2-propylpentanoyl, 4-methylpentanoyl,t-butylacetyl, 3-cyclohexylpropionyl, n-butanesulfonyl, benzylsulfonyl,4-methylbenzenesulfonyl, 2-naphthalenesulfonyl, 3-naphthalenesulfonyland 1-camphorsulfonyl.
 51. A method of claim 50, wherein k is 2 to 10.52. A method of claim 51, wherein k is 2 to
 5. 53. A method of claim 44,wherein R₇ is chemically removed from said third intermediate bytreating with titanium trichloride in MeOH, CH₂ Cl₂, or DMF.
 54. Amethod of claim 36, wherein R₇ is benzyloxycarbonyl.
 55. A method ofclaim 54, wherein R₆ is alkyl of 1 to about 12 carbon atoms.
 56. Amethod of claim 55, wherein R₆ is methyl, ethyl, propyl or isopropyl.57. A method of claim 56, wherein R₆ is ethyl.
 58. A method of claim 57,wherein R₅ is 4-methoxybenzyloxycarbonyl, t-butoxycarbonyl,t-amyloxycarbonyl, isobornyloxycarbonyl, adamantyloxycarbonyl and2-(4-biphenyl)-2-propyloxycarbonyl.
 59. A method of claim 58, whereinsaid R₅ removing reagent is a liquid mixture comprised of an acid andsolvent.
 60. A method of claim 59, wherein said acid is selected fromthe group consisting of HCl, trifluoroacetic acid and p-toluenesulfonicacid.
 61. A method of claim 60, wherein said acid is HCl and saidsolvent is ethanol.
 62. A method of claim 61, wherein said secondintermediate is coupled to said X-AA₁ -AA₂ . . . AA_(k) -OH using acoupling reagent selected from the group consisting of DCC with HOBt,EDC with HOBt, HBTU and TBTU.
 63. A method of claim 62, wherein R₇ ischemically removed from said third intermediate by treating withhydrogen gas in a liquid mixture comprised of catalyst, alcohol andacid.
 64. A method of claim 63, wherein said catalyst is palladium. 65.A method of claim 64, wherein said alcohol is ethanol and said acid isacetic acid.
 66. A method of claim 65, wherein said hydrolyzing reagentis an aqueous acid selected from the group consisting of HCl, HPF₆,methane sulfonic acid, perchloric acid, sulfuric acid, trifluoroaceticacid, trifluoromethane sulfonic acid and toluene sulfonic acid.
 67. Amethod of claim 66, wherein said aqueous acid is HPF₆ or HCl.
 68. Amethod of claim 67, wherein X is selected from a group consisting ofacetyl, 2-propylpentanoyl, 4-methylpentanoyl, t-butylacetyl,3-cyclohexylpropionyl, n-butanesulfonyl, benzylsulfonyl,4-methylbenzenesulfonyl, 2-naphthalenesulfonyl, 3-naphthalenesulfonyland 1-camphorsulfonyl.
 69. A method of claim 68, wherein k is 2 to 10.70. A method of claim 69, wherein k is 2 to
 5. 71. A method of claim 62,wherein R₇ is chemically removed from said third intermediate bytreating with titanium trichloride in MeOH, CH₂ Cl₂, or DMF.
 72. Amethod of claim 35, wherein R₇ is t-butoxycarbonyl.
 73. A method ofclaim 72, wherein R₆ is alkyl of 1 to about 12 carbon atoms.
 74. Amethod of claim 73, wherein R₆ is methyl, ethyl, propyl or isopropyl.75. A method of claim 74, wherein R₆ is ethyl.
 76. A method of claim 75,wherein R₅ is selected from the group consisting of benzyloxycarbonyl,isonicotinyloxycarbonyl and 2-chlorobenzyloxycarbonyl.
 77. A method ofclaim 76, wherein said R₅ removing reagent is hydrogen gas or a sourceof hydrogen gas in a liquid mixture comprised of catalyst and solvent.78. A method of claim 77 wherein said catalyst selected from the groupconsisting of platinum oxide or palladium.
 79. A method of claim 78,wherein said catalyst is palladium.
 80. A method of claim 79, whereinsaid solvent is comprised of ethanol and HCl.
 81. A method of claim 80,wherein said second intermediate is coupled to said X-AA₁ -AA₂ . . .AA_(k) -OH using a coupling reagent selected from the group consistingof DCC with HOBt, EDC with HOBt, HBTU and TBTU.
 82. A method of claim81, wherein said R₇ removing reagent is a liquid mixture comprised of anacid and solvent.
 83. A method of claim 82 wherein acid istrifluoroacetic acid and solvent is dichloromethane.
 84. A method ofclaim 83, wherein said hydrolyzing reagent is an aqueous acid selectedfrom the group consisting of HCl, HPF₆ methane sulfonic acid, perchloricacid, sulfuric acid, trifluoroacetic acid, trifluoromethane sulfonicacid and toluene sulfonic acid.
 85. A method of claim 84, wherein saidaqueous acid is HPF₆ or HCl.
 86. A method of claim 85, wherein X isselected from a group consisting of acetyl, 2-propylpentanoyl,4-methylpentanoyl, t-butylacetyl, 3-cyclohexylpropionyl,n-butanesulfonyl, benzylsulfonyl, 4-methylbenzenesulfonyl,2-naphthalenesulfonyl, 3-naphthalenesulfonyl and 1-camphorsulfonyl. 87.A method of claim 86, wherein k is 2 to
 10. 88. A method of claim 87,wherein k is 2 to
 5. 89. A method of claim 81, wherein R₇ is chemicallyremoved from said third intermediate by treating with titaniumtrichloride in MeOH, CH₂ Cl₂, or DMF.
 90. A method of claim 35, whereinR₇ is Fmoc.
 91. A method of claim 90, wherein R₆ is substituted with--C(O)--Y, wherein Y is O-polymeric support or NH-polymeric support. 92.A method of claim 91, wherein the polymeric support is selected from thegroup consisting of aminomethylated polystyrene resin, p-benzyloxybenzylalcohol resin, Merrifield resin, Rink amide resin, and MBHA resin.
 93. Amethod of claim 92, wherein the polymeric support is Merrifield resin orMBHA resin.
 94. A method of claim 91, wherein R₅ is hydrogen,4-methoxybenzyloxycarbonyl, t-butoxycarbonyl, t-amyloxycarbonyl,isobornyloxycarbonyl, adamantyloxycarbonyl or2-(4-biphenyl)-2-propyloxycarbonyl.
 95. A method of claim 94, wherein R₅is hydrogen.
 96. A method of claim 94, wherein R₅ is t-butoxycarbonyl.97. A compound of claim 1, wherein R₂ is substituted with --C(O)--Y,wherein Y is O-polymeric support or NH-polymeric support.
 98. A compoundof claim 97, wherein the polymeric support is selected from the groupconsisting of aminomethylated polystyrene resin, p-benzyloxybenzylalcohol resin, Merrifield resin, Rink amide resin, and MBHA resin.
 99. Acompound of claim 98, wherein the polymeric support is Merrifield resinor MBHA resin.
 100. A compound of claim 97, wherein R₁ is hydrogen,4-methoxybenzyloxycarbonyl, t-butoxycarbonyl, t-amyloxycarbonyl,isobornyloxycarbonyl, adamantyloxycarbonyl or2-(4biphenyl)-2-propyloxycarbonyl.
 101. A compound of claim 100, whereinR₁ is hydrogen.
 102. A compound of claim 100, wherein R₁ ist-butoxycarbonyl.
 103. A compound of the formula ##STR61## wherein R₅ isselected from the group consisting of benzyloxycarbonyl,isonicotinyloxycarbonyl, 2-chlorobenzyloxycarbonyl,4-methoxybenzyloxycarbonyl, t-butoxycarbonyl, t-amyloxycarbonyl,isobornyloxycarbonyl, adamantyloxycarbonyl,2-(4-biphenyl)-2-propyloxycarbonyl, 9-fluorenylmethoxycarbonyl andmethylsulfonylethoxycarbonyl;R₆ is selected from the group consisting ofalkyl of 1 to about 12 carbon atoms and aralkyl of about 7 to about 15carbon atoms, substituted with --C(O)--Y wherein Y is O-polymericsupport or NH-polymeric support; R₇ is selected from the groupconsisting of hydrogen, Fmoc, nitro, benzyloxycarbonyl, t-butoxycarbonyland adamantyloxycarbonyl; and R₈ is selected from the group consistingof hydrogen, alkyl of 1 to about 12 carbon atoms, aryl of about 6 toabout 14 carbon atoms, and aralkyl of about 7 to about 15 carbon atoms.104. A compound of claim 103, wherein the polymeric support is selectedfrom the group consisting of aminomethylated polystyrene resin,p-benzyloxybenzyl alcohol resin, Merrifield resin, Rink amide resin, andMBHA resin.
 105. A compound of claim 104, wherein the polymeric supportis Merrifield resin or MBHA resin.
 106. A compound of claim 103, whereinR₅ is hydrogen, 4-methoxybenzyloxycarbonyl, t-butoxycarbonyl,t-amyloxycarbonyl, isobornyloxycarbonyl, adamantyloxycarbonyl or2-(4-biphenyl)-2-propyloxycarbonyl.
 107. A compound of claim 106,wherein R₅ is hydrogen.
 108. A compound of claim 106, wherein R₅ ist-butoxycarbonyl.